Methyltransferase genes and uses thereof

ABSTRACT

The present invention relates to genes associated with the tocopherol biosynthesis pathway. More particularly, the present invention provides and includes nucleic acid molecules, proteins, and antibodies associated with genes that encode polypeptides that have methyltransferase activity. The present invention also provides methods for utilizing such agents, for example in gene isolation, gene analysis and the production of transgenic plants. Moreover, the present invention includes transgenic plants modified to express the aforementioned polypeptides. In addition, the present invention includes methods for the production of products from the tocopherol biosynthesis pathway.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S.provisional application No. 60/312,758, which is herein incorporated byreference in its entirety.

FIELD OF THE INVENTION

[0002] The present invention is in the field of plant genetics andbiochemistry. More specifically, the invention relates to genesassociated with the tocopherol biosynthesis pathway, namely thoseencoding methyltransferase activity, and uses of such genes.

BACKGROUND

[0003] Tocopherols are an important component of mammalian diets.Epidemiological evidence indicates that tocopherol supplementation canresult in decreased risk for cardiovascular disease and cancer, can aidin immune function, and is associated with prevention or retardation ofa number of degenerative disease processes in humans (Traber and Sies,Annu. Rev. Nutr. 16:321-347 (1996)). Tocopherol functions, in part, bystabilizing the lipid bilayer of biological membranes (Skrypin andKagan, Biochim. Biophys. Acta 815:209 (1995); Kagan, N.Y. Acad. Sci. p121, (1989); Gomez-Fernandez et al., Ann. N.Y Acad. Sci. p 109 (1989)),reducing polyunsaturated fatty acid (PUFA) free radicals generated bylipid oxidation (Fukuzawa et al., Lipids 17: 511-513 (1982)), andscavenging oxygen free radicals, lipid peroxy radicals andsinglet-oxygen species (Diplock et al. Ann. N Y Acad. Sci. 570: 72(1989); Fryer, Plant Cell Environ. 15(4):381-392 (1992)).

[0004] α-Tocopherol, often referred to as vitamin E, belongs to a classof lipid-soluble antioxidants that includes α, β, γ, and δ-tocopherolsand α, β, γ, and δ-tocotrienols. Although α, β, γ, and δ-tocopherols andα, β, γ, and δ-tocotrienols are sometimes referred to collectively as“vitamin E”, vitamin E is more appropriately defined chemically as αtocopherol. α-Tocopherol is significant for human health, in partbecause it is readily absorbed and retained by the body, and thereforehas a higher degree of bioactivity than other tocopherol species (Traberand Sies, Annu. Rev. Nutr. 16:321-347 (1996)). However, othertocopherols such as β, γ, and δ-tocopherols, also have significanthealth and nutritional benefits.

[0005] Tocopherols are primarily synthesized only by plants and certainother photosynthetic organisms, including cyanobacteria. As a result,mammalian dietary tocopherols are obtained almost exclusively from thesesources. Plant tissues vary considerably in total tocopherol content andtocopherol composition, with α-tocopherol the predominant tocopherolspecies found in green, photosynthetic plant tissues. Leaf tissue cancontain from 10-50 μg of total tocopherols per gram fresh weight, butmost of the world's major staple crops (e.g., rice, corn, wheat, potato)produce low to extremely low levels of total tocopherols, of which onlya small percentage is α-tocopherol (Hess, Vitamin E, α-tocopherol, InAntioxidants in Higher Plants, R. Alscher and J. Hess, Eds., CRC Press,Boca Raton. pp. 111-134 (1993)). Oil seed crops generally contain muchhigher levels of total tocopherols, but α-tocopherol is present only asa minor component in most oilseeds (Taylor and Barnes, Chem. Ind.,Oct.:722-726 (1981)).

[0006] The recommended daily dietary intake of 15-30 mg of vitamin E isquite difficult to achieve from the average American diet. For example,it would take over 750 grams of spinach leaves in which α-tocopherolcomprises 60% of total tocopherols, or 200-400 grams of soybean oil tosatisfy this recommended daily vitamin E intake. While it is possible toaugment the diet with supplements, most of these supplements containprimarily synthetic vitamin E, having eight stereoisomers, whereasnatural vitamin E is predominantly composed of only a single isomer.Furthermore, supplements tend to be relatively expensive, and thegeneral population is disinclined to take vitamin supplements on aregular basis. Therefore, there is a need in the art for compositionsand methods that either increase the total tocopherol production orincrease the relative percentage of α-tocopherol produced by plants.

[0007] In addition to the health benefits of tocopherols, increasedα-tocopherol levels in crops have been associated with enhancedstability and extended shelf life of plant products (Peterson,Cereal-Chem. 72(1):21-24 (1995); Ball, Fat-soluble vitamin assays infood analysis. A comprehensive review, London, Elsevier SciencePublishers Ltd. (1988)). Further, tocopherol supplementation of swine,beef, and poultry feeds has been shown to significantly increase meatquality and extend the shelf life of post-processed meat products byretarding post-processing lipid oxidation, which contributes to theundesirable flavor components (Sante and Lacourt, J. Sci. Food Agric.65(4):503-507 (1994); Buckley et al., J. of Animal Science 73:3122-3130(1995)).

[0008] Tocopherol Biosynthesis

[0009] The plastids of higher plants exhibit interconnected biochemicalpathways leading to secondary metabolites including tocopherols. Thetocopherol biosynthetic pathway in higher plants involves condensationof homogentisic acid and phytylpyrophosphate to form 2-methyl-6phytylplastoquinol (Fiedler et al., Planta 155: 511-515 (1982); Soll etal., Arch. Biochem. Biophys. 204: 544-550 (1980); Marshall et al.,Phytochem. 24: 1705-1711 (1985)). This plant tocopherol pathway can bedivided into four parts: 1) synthesis of homogentisic acid, whichcontributes to the aromatic ring of tocopherol; 2) synthesis ofphytylpyrophosphate, which contributes to the side chain of tocopherol;3) joining of HGA and phytylpyrophosphate via a prenyltransferasefollowed by a subsequent cyclization; 4) and S-adenosyl methioninedependent methylation of an aromatic ring, which affects the relativeabundance of each of the tocopherol species.

[0010] Synthesis of Homogentisic Acid

[0011] Homogentisic acid is the common precursor to both tocopherols andplastoquinones. In at least some bacteria the synthesis of homogentisicacid is reported to occur via the conversion of chorismate to prephenateand then to p-hydroxyphenylpyruvate via a bifunctional prephenatedehydrogenase. Examples of bifunctional bacterial prephenatedehydrogenase enzymes include the proteins encoded by the tyrA genes ofErwinia herbicola and Escherichia coli. The tyrA gene product catalyzesthe production of prephenate from chorismate, as well as the subsequentdehydrogenation of prephenate to form p-hydroxyphenylpyruvate (p-HPP),the immediate precursor to homogentisic acid. p-HPP is then converted tohomogentisic acid by hydroxyphenylpyruvate dioxygenase (HPPD). Incontrast, plants are believed to lack prephenate dehydrogenase activity,and it is generally believed that the synthesis of homogentisic acidfrom chorismate occurs via the synthesis and conversion of theintermediate arogenate. Since pathways involved in homogentisic acidsynthesis are also responsible for tyrosine formation, any alterationsin these pathways can also result in the alteration in tyrosinesynthesis and the synthesis of other aromatic amino acids.

[0012] Synthesis of Phytylpyrophosphate

[0013] Tocopherols are a member of the class of compounds referred to asthe isoprenoids. Other isoprenoids include carotenoids, gibberellins,terpenes, chlorophyll and abscisic acid. A central intermediate in theproduction of isoprenoids is isopentenyl diphosphate (IPP). Cytoplasmicand plastid-based pathways to generate IPP have been reported. Thecytoplasmic based pathway involves the enzymes acetoacetyl CoA thiolase,HMGCoA synthase, HMGCoA reductase, mevalonate kinase, phosphomevalonatekinase, and mevalonate pyrophosphate decarboxylase.

[0014] Recently, evidence for the existence of an alternative, plastidbased, isoprenoid biosynthetic pathway emerged from studies in theresearch groups of Rohmer and Arigoni (Eisenreich et al., Chem. Bio.,5:R221-R233 (1998); Rohmer, Prog. Drug. Res., 50:135-154 (1998); Rohmer,Comprehensive Natural Products Chemistry, Vol. 2, pp. 45-68, Barton andNakanishi (eds.), Pergamon Press, Oxford, England (1999)), who foundthat the isotope labeling patterns observed in studies on certaineubacterial and plant terpenoids could not be explained in terms of themevalonate pathway. Arigoni and coworkers subsequently showed that1-deoxyxylulose, or a derivative thereof, serves as an intermediate ofthe novel pathway, now referred to as the MEP pathway (Rohmer et al.,Biochem. J., 295:517-524 (1993); Schwarz, Ph.D. thesis, EidgenössicheTechnische Hochschule, Zurich, Switzerland (1994)). Recent studiesshowed the formation of 1-deoxyxylulose 5-phosphate (Broers, Ph.D.thesis (Eidgenössiche Technische Hochschule, Zurich, Switzerland)(1994)) from one molecule each of glyceraldehyde 3-phosphate (Rohmer,Comprehensive Natural Products Chemistry, Vol. 2, pp. 45-68, Barton andNakanishi, eds., Pergamon Press, Oxford, England (1999)) and pyruvate(Eisenreich et al., Chem. Biol., 5:R223-R233 (1998); Schwarz supra;Rohmer et al., J. Am. Chem. Soc., 118:2564-2566 (1996); and Sprenger etal., Proc. Natl. Acad. Sci. USA, 94:12857-12862 (1997)) by an enzymeencoded by the dxs gene (Lois et al., Proc. Natl. Acad. Sci. USA,95:2105-2110 (1997); and Lange et al., Proc. Natl. Acad. Sci. USA,95:2100-2104 (1998)). 1-Deoxyxylulose 5-phosphate can be furtherconverted into 2-C-methylerythritol 4-phosphate (Arigoni et al., Proc.Natl. Acad. Sci. USA, 94:10600-10605 (1997)) by a reductoisomeraseencoded by the dxr gene (Bouvier et al., Plant Physiol, 11 7:1421-1431(1998); and Rohdich et al., Proc. Natl. Acad. Sci. USA, 96:11758-11763(1999)).

[0015] Reported genes in the MEP pathway also include ygbP, whichcatalyzes the conversion of 2-C-methylerythritol 4-phosphate into itsrespective cytidyl pyrophosphate derivative and ygbB, which catalyzesthe conversion of 4-phosphocytidyl-2C-methyl-D-erythritol into2C-methyl-D-erythritol, 3,4-cyclophosphate. These genes are tightlylinked on the E. coli genome (Herz et al., Proc. Natl. Acad. Sci.U.S.A., 97(6):2485-2490 (2000)).

[0016] Once IPP is formed by the MEP pathway, it is converted to GGDP byGGDP synthase, and then to phytylpyrophosphate, which is the centralconstituent of the tocopherol side chain.

[0017] Combination and Cyclization

[0018] Homogentisic acid is combined with either phytyl-pyrophosphate orsolanyl-pyrophosphate by phytyl/prenyl transferase forming2-methyl-6-phytyl plastoquinol or 2-methyl-6-solanyl plastoquinol,respectively. 2-methyl-6-solanyl plastoquinol is a precursor to thebiosynthesis of plastoquinones, while 2-methyl-6-phytyl plastoquinol isultimately converted to tocopherol.

[0019] Methylation of the Aromatic Ring

[0020] The major structural difference between each of the tocopherolsubtypes is the position of the methyl groups around the phenyl ring.Both 2-methyl-6-phytyl plastoquinol and 2-methyl-6-solanyl plastoquinolserve as substrates for2-methyl-6-phytylplastoquinol/2-methyl-6-solanylplastoquinol-9methyltransferase (Methyl Transferase 1; MT1), which catalyzes theformation of plastoquinol-9 and γ-tocopherol respectively, bymethylation of the 7 position. Subsequent methylation at the 5 positionof γ-tocopherol by γ-tocopherol methyl-transferase (GMT) generates thebiologically active α-tocopherol. Additional potential MT1 substratesinclude 2-methyl-5-phytylplastoquinol and 2-methyl-3-phytylplastoquinol.Additional potential substrates for GMT include δ-tocopherol and γ- andδ-tocotrienol.

[0021] There is a need in the art for nucleic acid molecules encodingenzymes involved in tocopherol biosynthesis, as well as related enzymesand antibodies for the enhancement or alteration of tocopherolproduction in plants. There is a further need for transgenic organismsexpressing those nucleic acid molecules involved in tocopherolbiosynthesis, which are capable of nutritionally enhancing food and feedsources.

SUMMARY OF THE INVENTION

[0022] The present invention includes and provides a substantiallypurified nucleic acid molecule comprising a nucleic acid sequenceselected from the group consisting of SEQ ID NOs: 2-17, 50, and 85.

[0023] The present invention includes and provides a substantiallypurified nucleic acid molecule comprising a nucleic acid sequence thatencodes an amino acid sequence selected from the group consisting of SEQID NO: 19-31 and 33-38.

[0024] The present invention includes and provides a substantiallypurified nucleic acid molecule comprising as operably linked components:(A) a promoter region which functions in a plant cell to cause theproduction of an mRNA molecule; (B) a heterologous nucleic acid moleculeencoding a polypeptide molecule comprising a sequence selected from thegroup consisting of SEQ ID NOs: 19-31, 33-41.

[0025] The present invention includes and provides a substantiallypurified protein comprising an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 19-31, and 33-38.

[0026] The present invention includes and provides an antibody capableof specifically binding a substantially purified protein comprising anamino acid sequence selected from the group consisting of SEQ ID NOs:19-31, and 33-38.

[0027] The present invention includes and provides a transformed planthaving an exogenous nucleic acid molecule that encodes a polypeptidemolecule comprising a sequence selected from the group consisting of SEQID NOs: 19-31, and 33-41.

[0028] The present invention includes and provides a transformed planthaving an exogenous nucleic acid molecule that encodes a polypeptidemolecule comprising a sequence selected from the group consisting of SEQID NOs: 46-49.

[0029] The present invention includes and provides a method for reducingexpression of MT1 or GMT in a plant comprising: (A) transforming a plantwith a nucleic acid molecule, said nucleic acid molecule having anexogenous promoter region which functions in plant cells to cause theproduction of a mRNA molecule, wherein said exogenous promoter region islinked to a transcribed nucleic acid molecule having a transcribedstrand and a non-transcribed strand, wherein said transcribed strand iscomplementary to a nucleic acid molecule comprising a nucleic acidsequence selected from the group consisting of SEQ ID NOs: 2-17, 50, and85; and wherein said transcribed nucleic acid molecule is linked to a 3′non-translated sequence that functions in the plant cells to causetermination of transcription and addition of polyadenylatedribonucleotides to a 3′ end of the mRNA sequence; and (B) growing saidtransformed plant.

[0030] The present invention includes and provides a transformed plantcomprising a nucleic acid molecule comprising an exogenous promoterregion which functions in plant cells to cause the production of a mRNAmolecule, wherein the exogenous promoter region is linked to atranscribed nucleic acid molecule having a transcribed strand and anon-transcribed strand, wherein the transcribed strand is complementaryto a nucleic acid molecule comprising a nucleic acid sequence selectedfrom the group consisting of SEQ ID NOs: 2-17, 50, 85, and wherein thetranscribed nucleic acid molecule is linked to a 3′ non-translatedsequence that functions in the plant cells to cause termination oftranscription and addition of polyadenylated ribonucleotides to a 3′ endof the mRNA sequence; wherein, the expression of MT1, GMT or both isreduced relative to a plant with a similar genetic background butlacking the exogenous nucleic acid molecule.

[0031] The present invention includes and provides method for increasingthe γ-tocopherol content in a plant comprising: (A) transforming a plantwith a nucleic acid molecule, the nucleic acid molecule comprising anexogenous promoter region which functions in plant cells to cause theproduction of a mRNA molecule, wherein the exogenous promoter region islinked to a transcribed nucleic acid molecule comprising a transcribedstrand and a non-transcribed strand, wherein the transcribed strand iscomplementary to a nucleic acid molecule comprising a nucleic acidsequence selected from the group consisting of SEQ ID NOs: 2-17, 50, and85; and wherein the transcribed nucleic acid molecule is linked to a 3′non-translated sequence that functions in the plant cells to causetermination of transcription and addition of polyadenylatedribonucleotides to a 3′ end of the mRNA sequence; and (C) growing thetransformed plant.

[0032] The current invention further includes and provides a transformedplant comprising: (A) a first nucleic acid molecule comprising anexogenous promoter region which functions in plant cells to cause theproduction of a mRNA molecule, wherein the exogenous promoter region islinked to a transcribed nucleic acid molecule having a transcribedstrand and a non-transcribed strand, wherein the transcribed strand iscomplementary to a nucleic acid molecule comprising a nucleic acidsequence selected from the group consisting of SEQ ID NOs: 2-17, 50, and85, and wherein the transcribed nucleic acid molecule is linked to a 3′non-translated sequence that functions in the plant cells to causetermination of transcription and addition of polyadenylatedribonucleotides to a 3′ end of the mRNA sequence; and (B) a secondnucleic acid molecule comprising an exogenous promoter region whichfunctions in plant cells to cause the production of a mRNA molecule,wherein the exogenous promoter region is linked to a nucleic acidmolecule comprising a sequence selected from the group consisting of SEQID NOs: 42-45, wherein the γ-tocopherol content of the transformed plantis increased relative to a plant with a similar genetic background butlacking the exogenous nucleic acid molecule.

[0033] The present invention includes and provides a method of producinga plant having a seed with an increased α-tocopherol level comprising:(A) transforming the plant with a nucleic acid molecule, wherein thenucleic acid molecule comprises a sequence encoding a polypeptidemolecule comprising a sequence selected from the group consisting of SEQID NOs: 19-31, 33-38, and 39-41; and (B) growing the transformed plant.

[0034] The present invention includes and provides a method of producinga plant having a seed with an increased γ-tocopherol level comprising:(A) transforming the plant with a nucleic acid molecule, wherein thenucleic acid molecule comprises a nucleic acid sequence that encodes apolypeptide molecule comprising a sequence selected from the groupconsisting of SEQ ID NOs: 46-49; and (B) growing the transformed plant.

[0035] The present invention includes and provides a method ofaccumulating α-tocopherol in a seed comprising: (A) growing a plant witha heterologous nucleic acid molecule, wherein the heterologous nucleicacid molecule comprises a sequence encoding a polypeptide moleculecomprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 19-31, 33-38, and 39-41; and (B) isolating said seed fromsaid plant with a heterologous nucleic acid molecule.

[0036] The present invention includes and provides a method ofaccumulating γ-tocopherol in a seed comprising: (A) growing a plant witha heterologous nucleic acid molecule, wherein the heterologous nucleicacid molecule comprises a sequence encoding a polypeptide moleculecomprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 46-49; and (B) isolating said seed from said plant with aheterologous nucleic acid molecule.

[0037] The present invention includes and provides a seed derived from atransformed plant having an exogenous nucleic acid molecule comprising anucleic acid sequence encoding an polypeptide molecule comprising asequence selected from the group consisting of SEQ ID NOs: 19-31, 33-38,and 39-41, wherein the seed has an increased α-tocopherol level relativeto seeds from a plant having a similar genetic background but lackingthe exogenous nucleic acid molecule.

[0038] The present invention includes and provides an oil derived from aseed of a transformed plant, wherein the transformed plant contains anexogenous nucleic acid molecule comprising a nucleic acid sequenceencoding a polypeptide molecule comprising a sequence selected from thegroup consisting of SEQ ID NOs: 19-31, 33-38, and 39-41.

[0039] The present invention includes and provides feedstock comprisinga transformed plant or part thereof, wherein the transformed plant hasan exogenous nucleic acid molecule that comprises a nucleic acidsequence encoding a polypeptide molecule comprising a sequence selectedfrom the group consisting of SEQ ID NOs: 19-31, 33-38, and 39-41.

[0040] The present invention includes and provides a meal comprisingplant material manufactured from a transformed plant, wherein thetransformed plant has an exogenous nucleic acid molecule that comprisesa nucleic acid sequence encoding a polypeptide molecule comprising asequence selected from the group consisting of SEQ ID NOs: 19-31, 33-38,and 39-41.

[0041] The present invention includes and provides a seed derived from atransformed plant having an exogenous nucleic acid molecule comprising asequence encoding a polypeptide molecule comprising a sequence selectedfrom the group consisting of SEQ ID NOs: 46-49, wherein the seed has anincreased tocopherol level relative to seeds from a plant having asimilar genetic background but lacking the exogenous nucleic acidmolecule.

[0042] The present invention includes and provides oil derived from aseed of a transformed plant, wherein the transformed plant contains anexogenous nucleic acid molecule comprising a nucleic acid sequenceencoding a polypeptide molecule comprising a sequence selected from thegroup consisting of SEQ ID NOs: 46-49.

[0043] The present invention also includes and provides feedstockcomprising a transformed plant or part thereof, wherein the transformedplant has an exogenous nucleic acid molecule that comprises a nucleicacid sequence encoding a polypeptide molecule comprising a sequenceselected from the group consisting of SEQ ID NOs: 46-49.

[0044] The present invention also includes and provides meal comprisingplant material manufactured from a transformed plant, wherein thetransformed plant has an exogenous nucleic acid molecule that comprisesa nucleic acid sequence encoding a polypeptide molecule comprising asequence selected from the group consisting of SEQ ID NO: 46-49.

[0045] The present invention also includes and provides a host cellcomprising a nucleic acid molecule comprising a nucleic acid sequenceselected from the group consisting of SEQ ID NOs: 2 -17, 42-45 andcomplements thereof.

[0046] The present invention also includes and provides an introducedfirst nucleic acid molecule that encodes a polypeptide moleculecomprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 19-31, 33-38, and 39-41, and an introduced second nucleicacid molecule encoding an enzyme selected from the group consisting oftyrA, slr1736, ATPT2, dxs, dxr, GGPPS, HPPD, GMT, MT1, tMT2, AANT1, slr1737, and an antisense construct for homogentisic acid dioxygenase.

[0047] The present invention also includes and provides a transformedplant comprising an introduced first nucleic acid molecule that encodesa polypeptide molecule comprising an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 46-49, and an introduced secondnucleic acid molecule encoding an enzyme selected from the groupconsisting of tyrA, slr1736, ATPT2, dxs, dxr, GGPPS, HPPD, GMT, MT1,tMT2, AANT1, sir 1737, and an antisense construct for homogentisic aciddioxygenase.

[0048] The present invention also includes and provides a plantcomprising an introduced nucleic acid molecule comprising a nucleic acidsequence selected from the group consisting of SEQ ID NOs: 42-45,wherein said transformed plant produces a seed having increased totaltocopherol relative to seed of a plant with a similar genetic backgroundbut lacking said introduced nucleic acid molecule.

[0049] The present invention also includes and provides a plantcomprising an introduced nucleic acid molecule comprising a nucleic acidsequence selected from the group consisting of SEQ ID NOs: 2-17, 50, 85,wherein said transformed plant produces a seed having increased totaltocopherol relative to seed of a plant with a similar genetic backgroundbut lacking said introduced nucleic acid molecule.

[0050] The present invention also includes and provides a plantcomprising a first introduced nucleic acid molecule comprising a nucleicacid sequence selected from the group consisting of SEQ ID NOs: 2-17,50, and 85 and a second introduced nucleic acid molecule comprising anucleic acid sequence selected from the group consisting of SEQ ID NOs:42-45, wherein said transformed plant produces a seed having increasedtotal tocopherol relative to seed of a plant with a similar geneticbackground but lacking both said introduced first nucleic acid moleculeand said introduced second nucleic acid molecule.

DESCRIPTION OF THE NUCLEIC AND AMINO ACID SEQUENCES

[0051] SEQ ID NO: 1 sets forth a nucleic acid sequence of a DNA moleculethat encodes an Arabidopsis thaliana gamma-tocopherol methyltransferase.

[0052] SEQ ID NO: 2 sets forth a nucleic acid sequence of a DNA moleculethat encodes an Arabidopsis thaliana, Columbia ecotype, gamma-tocopherolmethyltransferase.

[0053] SEQ ID NO: 3 sets forth a nucleic acid sequence of a DNA moleculethat encodes an Oryza sativa gamma-tocopherol methyltransferase.

[0054] SEQ ID NO: 4 sets forth a nucleic acid sequence of a DNA moleculethat encodes a Gossypium hirsutum gamma-tocopherol methyltransferase.

[0055] SEQ ID NO: 5 sets forth a nucleic acid sequence of a DNA moleculethat encodes a Cuphea pulcherrima gamma-tocopherol methyltransferase.

[0056] SEQ ID NO: 6 sets forth a nucleic acid sequence of a DNA moleculethat encodes a Brassica napus S8 gamma-tocopherol methyltransferase.

[0057] SEQ ID NO: 7 sets forth a nucleic acid sequence of a DNA moleculethat encodes a Brassica napus P4 gamma-tocopherol methyltransferase.

[0058] SEQ ID NO: 8 sets forth a nucleic acid sequence of a DNA moleculethat encodes a Brassica napus S8 gamma-tocopherol methyltransferase.

[0059] SEQ ID NO: 9 sets forth a nucleic acid sequence of a DNA moleculethat encodes a Brassica napus P4 gamma-tocopherol methyltransferase.

[0060] SEQ ID NO: 10 sets forth a nucleic acid sequence of a DNAmolecule that encodes a Lycopersicon esculentum gamma-tocopherolmethyltransferase.

[0061] SEQ ID NO: 11 sets forth a nucleic acid sequence of a DNAmolecule that encodes a Glycine max gamma-tocopherol methyltransferase1.

[0062] SEQ ID NO: 12 sets forth a nucleic acid sequence of a DNAmolecule that encodes a Glycine max gamma-tocopherol methyltransferase2.

[0063] SEQ ID NO: 13 sets forth a nucleic acid sequence of a DNAmolecule that encodes a Glycine max gamma-tocopherol methyltransferase3.

[0064] SEQ ID NO: 14 sets forth a nucleic acid sequence of a DNAmolecule that encodes a Tagetes erecta gamma-tocopherolmethyltransferase.

[0065] SEQ ID NO: 15 sets forth a nucleic acid sequence of a DNAmolecule that encodes a Sorghum bicolor gamma-tocopherolmethyltransferase.

[0066] SEQ ID NO: 16 sets forth a nucleic acid sequence of a DNAmolecule that encodes a Nostoc punctiforme gamma-tocopherolmethyltransferase.

[0067] SEQ ID NO: 17 sets forth a nucleic acid sequence of a DNAmolecule that encodes an Anabaena gamma-tocopherol methyltransferase.

[0068] SEQ ID NO: 18 set forth a derived amino acid sequence of anArabidopsis thaliana gamma-tocopherol methyltransferase.

[0069] SEQ ID NO: 19 sets forth a derived amino acid sequence of anArabidopsis thaliana, Columbia ecotype, gamma-tocopherolmethyltransferase.

[0070] SEQ ID NO: 20 sets forth a derived amino acid sequence of anOryza sativa gamma-tocopherol methyltransferase.

[0071] SEQ ID NO: 21 sets forth a derived amino acid sequence of a Zeamays gamma-tocopherol methyltransferase.

[0072] SEQ ID NO: 22 sets forth a derived amino acid sequence of aGossypium hirsutum gamma-tocopherol methyltransferase.

[0073] SEQ ID NO: 23 sets forth a derived amino acid sequence of aCuphea pulcherrima gamma-tocopherol methyltransferase.

[0074] SEQ ID NO: 24 sets forth a derived amino acid sequence of aBrassica napus S8 gamma-tocopherol methyltransferase.

[0075] SEQ ID NO: 25 sets forth a derived amino acid sequence of aBrassica napus P4 gamma-tocopherol methyltransferase.

[0076] SEQ ID NO: 26 sets forth a derived amino acid sequence of aLycopersicon esculentum gamma-tocopherol methyltransferase.

[0077] SEQ ID NO: 27 sets forth a derived amino acid sequence of aGlycine max gamma-tocopherol methyltransferase.

[0078] SEQ ID NO: 28 sets forth a derived amino acid sequence of aGlycine max gamma-tocopherol methyltransferase.

[0079] SEQ ID NO: 29 sets forth a derived amino acid sequence of aGlycine max gamma-tocopherol methyltransferase.

[0080] SEQ ID NO: 30 sets forth a derived amino acid sequence of aTagetes erecta gamma-tocopherol methyltransferase.

[0081] SEQ ID NO: 31 sets forth a derived amino acid sequence of aSorghum bicolor gamma-tocopherol methyltransferase.

[0082] SEQ ID NO: 32 sets forth an amino acid sequence of a pea rubiscosmall subunit chloroplast targeting sequence (CTP1).

[0083] SEQ ID NO: 33 sets forth a derived mature amino acid sequence ofa Brassica napus S8 gamma-tocopherol methyltransferase.

[0084] SEQ ID NO: 34 sets forth a derived mature amino acid sequence ofa Brassica napus P4 gamma-tocopherol methyltransferase.

[0085] SEQ ID NO: 35 sets forth a derived mature amino acid sequence ofa Cuphea pulcherrima gamma-tocopherol methyltransferase.

[0086] SEQ ID NO: 36 sets forth a derived mature amino acid sequence ofa Gossypium hirsutum gamma-tocopherol methyltransferase.

[0087] SEQ ID NO: 37 sets forth a derived mature amino acid sequence ofa Tagetes erecta gamma-tocopherol methyltransferase.

[0088] SEQ ID NO: 38 sets forth a derived mature amino acid sequence ofa Zea mays gamma-tocopherol methyltransferase.

[0089] SEQ ID NO: 39 sets forth a derived amino acid sequence of aNostoc punctiforme gamma-tocopherol methyltransferase.

[0090] SEQ ID NO: 40 sets forth a derived amino acid sequence of anAnabaena gamma-tocopherol methyltransferase.

[0091] SEQ ID NO: 41 sets forth an amino acid sequence of Synechocystisgamma-tocopherol methyltransferase.

[0092] SEQ ID NO: 42 sets forth a nucleic acid sequence of a nucleicacid molecule encoding a Synechocystis pcc 68032-methyl-6-phytylplastoquinol/2-methyl-6-solanylplastoquinol-9methyltransferase.

[0093] SEQ ID NO: 43 sets forth a nucleic acid sequence of a nucleicacid molecule encoding an Anabaena2-methyl-6-phytylplastoquinol/2-methyl-6-solanylplastoquinol-9methyltransferase.

[0094] SEQ ID NO: 44 sets forth a nucleic acid sequence of a nucleicacid molecule encoding a Synechococcus2-methyl-6-phytylplastoquinol/2-methyl-6-solanylplastoquinol-9methyltransferase.

[0095] SEQ ID NO: 45 sets forth a nucleic acid sequence of a nucleicacid molecule encoding a Prochlorococcus marinus2-methyl-6-phytylplastoquinol/2-methyl-6-solanylplastoquinol-9methyltransferase.

[0096] SEQ ID NO: 46 sets forth a derived amino acid sequence of anSynechocystis pcc 68032-methyl-6-phytylplastoquinol/2-methyl-6-solanylplastoquinol-9methyltransferase.

[0097] SEQ ID NO: 47 sets forth a derived amino acid sequence of anAnabaena 2-methyl-6-phytylplastoquinol/2-methyl-6-solanylplastoquinol-9methyltransferase.

[0098] SEQ ID NO: 48 sets forth a derived amino acid sequence of aSynechococcus2-methyl-6-phytylplastoquinol/2-methyl-6-solanylplastoquinol-9methyltransferase.

[0099] SEQ ID NO: 49 sets forth a derived amino acid sequence of aProchlorococcus2-methyl-6-phytylplastoquinol/2-methyl-6-solanylplastoquinol-9methyltransferase.

[0100] SEQ ID NO: 50 sets forth a nucleic acid sequence of an Oryzasativa gamma-tocopherol methyltransferase.

[0101] SEQ ID NOs: 51 and 52 set forth a nucleic acid sequence ofprimers for use in amplifying a Brassica napus S8 gamma methyltransferase.

[0102] SEQ ID NOs: 53 and 54 set forth a nucleic acid sequence ofprimers for use in amplifying a Brassica napus P4 gamma methyltransferase.

[0103] SEQ ID NOs: 55 and 56 set forth a nucleic acid sequence ofprimers for use in amplifying a Cuphea pulcherrima gamma methyltransferase.

[0104] SEQ ID NOs: 57 and 58 set forth a nucleic acid sequence ofprimers for use in amplifying a Gossypium hirsutum gamma methyltransferase.

[0105] SEQ ID NOs: 59 and 60 set forth a nucleic acid sequence ofprimers for use in amplifying a mature Brassica napus S8 gamma methyltransferase and a mature Brassica napus P4 gamma methyl transferase.

[0106] SEQ ID NOs: 61 and 62 set forth a nucleic acid sequence ofprimers for use in amplifying a mature Cuphea pulcherrima gamma methyltransferase.

[0107] SEQ ID NOs: 63 and 64 set forth a nucleic acid sequence ofprimers for use in amplifying a mature Gossypium hirsutum gamma methyltransferase.

[0108] SEQ ID NOs: 65 and 66 set forth a nucleic acid sequence ofprimers for use in amplifying a mature Tagetes erecta gamma methyltransferase.

[0109] SEQ ID NOs: 67 and 68 set forth a nucleic acid sequence ofprimers for use in amplifying a Nostoc punctiforme gamma methyltransferase.

[0110] SEQ ID NOs: 69 and 70 set forth a nucleic acid sequence ofprimers for use in amplifying an Anabaena gamma methyl transferase.

[0111] SEQ ID NOs: 71 and 72 set forth a nucleic acid sequence ofprimers for use in amplifying an Anabaena2-methyl-6-phytylplastoquinol/2-methyl-6-solanylplastoquinol-9methyltransferase.

[0112] SEQ ID NOs: 73 and 74 set forth a nucleic acid sequence ofprimers for use in amplifying a mature Zea mays gamma methyltransferase.

[0113] SEQ ID NOs: 75 and 76 set forth a nucleic acid sequence ofprimers for use in amplifying an Arabidopsis gamma methyl transferase.

[0114] SEQ ID NOs: 77 and 78 set forth a nucleic acid sequence ofprimers for use in amplifying an Arabidopsis gamma methyl transferase.

[0115] SEQ ID NOs: 79 and 80 set forth a nucleic acid sequence ofprimers for use in amplifying an Arcelin 5 promoter.

[0116] SEQ ID NO: 81 sets forth a 5′ translational start region of anucleic acid sequence corresponding to an Arcelin 5 promoter frompARC5-1.

[0117] SEQ ID NO: 82 sets forth a 5′ translational start region of anucleic acid sequence corresponding to an Arcelin 5 promoter frompARC5-1M.

[0118] SEQ ID NOs: 83 and 84 set forth nucleic acid sequences of primersfor use in amplifying an Anabaena putative-MT1 coding sequence.

[0119] SEQ ID NO: 85 sets forth a nucleic acid sequence of a Zea maysgamma-tocopherol methyltransferase.

BRIEF DESCRIPTION OF THE FIGURES

[0120]FIG. 1 is a schematic of construct pET-DEST42.

[0121]FIG. 2 is a schematic of construct pCGN9979.

[0122]FIG. 3 is a schematic of construct pMON26592.

[0123]FIG. 4 is a schematic of construct pMON26593.

[0124]FIG. 5 is a schematic of construct pMON55524.

[0125]FIG. 6 is a schematic of construct pMON36500.

[0126]FIG. 7 is a schematic of construct pMON36501.

[0127]FIG. 8 is a schematic of construct pMON36502.

[0128]FIG. 9 is a schematic of construct pMON36503.

[0129]FIG. 10 is a schematic of construct pMON36504.

[0130]FIG. 11 is a schematic of construct pMON36505.

[0131]FIG. 12 is a schematic of construct pMON36506.

[0132]FIG. 13 is a schematic of construct pMON67157.

[0133]FIG. 14 is a graph depicting the soy seed tocopherol content andcomposition from pooled seed of the R1 generation of plants transformedwith pMON36503. This construct expresses an A. thaliana GMT under p7Spromoter control.

[0134]FIG. 15 is a graph depicting the soy seed tocopherol content andcomposition from pooled seed of the R1 generation of plants transformedwith pMON36505. This construct expresses an A. thaliana GMT underarcelin5 promoter control.

[0135]FIG. 16 is a graph depicting the soy seed tocopherol content andcomposition from pooled seed of the R1 generation of plants transformedwith pMON36506. This construct expresses an A. thaliana GMT under thecontrol of the modified arcelin 5 promoter.

[0136]FIG. 17 is a graph representing the enzyme activities of variousgamma-methyltransferases (GMT) and a tocopherol methyl transferase 1(MT1) in recombinant E. coli crude extract preparations. Enzymeactivities are expressed as either pmol α-tocopherol (GMT) or2,3-dimethyl-5-phytylplastoquinol (MT1) formation per mg protein permin. Vector designations stand for the following recombinant genes:pMON67171, mature cotton GMT; pMON67173, mature Cuphea pulcherrima GMT;pMON67177, mature marigold GMT; pMON67181, mature Brassica napus S8 GMT;pMON67183, Zea mays GMT; pMON67175, Anabaena GMT; pMON67176, Nostoc GMT;and pMON67174, Anabaena MT1.

[0137]FIG. 18 is an HPLC chromatogram, representing themethyltransferase activity of recombinant expressed Anabaenamethyltransferase 1. Enzyme activity is monitored on crude cell extractsfrom E. coli harboring pMON67174.

[0138]FIG. 19 is an HPLC chromatogram, representing theMethyltransferase activity of recombinant expressed Anabaenamethyltransferase 1 without 2-methylphytylplastoquinol substrate(negative control). Enzyme activity is monitored on crude cell extractsfrom E. coli harboring pMON67174.

[0139]FIG. 20 is an HPLC chromatogram, representing themethyltransferase 1 activity in isolated pea chloroplasts (positivecontrol).

[0140]FIGS. 21A and 21B are graphs representing the α and γ-tocopherollevels in Arabidopsis T₂ seed from 5 transgenic control plantscontaining the napin binary vector (9979), 15 transgenic plantsexpressing the Arabidopsis thaliana GMT gene (Columbia ecotype) underthe control of the napin promoter (67156) and 13 transgenic plantsexpressing the Brassica napus P4 GMT under the control of the napinpromoter (67159).

[0141]FIGS. 22A and 22B are graphs representing the α and γ-tocopherollevels in Arabidopsis T₂ seeds from 5 transgenic plants containing thenapin binary vector (9979), 15 transgenic plants expressing the Cupheapulcherrima GMT gene under the control of the napin promoter (67158) and1 transgenic plant expressing the Brassica napus P4 GMT under thecontrol of the napin promoter (67159).

[0142]FIG. 23 is a graph representing the average seed γ-tocopherollevel in transformed Arabidopsis plants harboring expression constructsfor the Arabidopsis thaliana ecotype Columbia GMT (67156), the cupheaGMT (67158), the Brassica P4 GMT (67159), the cotton GMT (67160), andthe Brassica S8 GMT (67170).

[0143]FIG. 24 is a graph representing the average seed α-tocopherollevel in transformed Brassica plants.

[0144]FIG. 25 shows the percent of seed δ-tocopherol in Arabidopsis T2seed from lines expressing MT1 under the control of the napin promoter.

[0145]FIG. 26 shows T₃ seed δ-tocopherol levels in two lines expressingMT1 under the control of the napin promoter.

[0146]FIG. 27 represents pMON67212.

[0147]FIG. 28 represents pMON67213.

[0148]FIG. 29 shows total tocopherol level for Arabidopsis transformedwith an MT1 and prenyltransferase double construct.

[0149]FIG. 30 shows γ tocopherol level for Arabidopsis transformed withan MT1 and prenyltransferase double construct.

[0150]FIG. 31 shows γ-tocopherol level for Arabidopsis transformed withan MT1 and prenyltransferase double construct.

[0151]FIG. 32 shows α-tocopherol level for Arabidopsis transformed withan MT1 and prenyltransferase double construct.

[0152]FIG. 33 is a graph showing 2-Methylphytylplastoquinolmethyltransferase activity obtained with recombinant proteins and a peachloroplast control. Data are obtained with recombinant proteins frommicrobial and plant sources.

[0153]FIG. 34 is a graph showing GMT substrate specificity forgamma-tocopherols versus gamma-tocotrienols. GMT activity is measuredwith recombinant expressed gamma methyltransferases from cotton,Anabaena, and corn, using gamma tocopherol or gamma-tocotrienol andS-adenosylmethionine as a substrate.

DETAILED DESCRIPTION

[0154] The present invention provides a number of agents, for example,nucleic acid molecules and polypeptides associated with the synthesis oftocopherol, and provides uses of such agents.

[0155] Agents

[0156] The agents of the invention will preferably be “biologicallyactive” with respect to either a structural attribute, such as thecapacity of a nucleic acid to hybridize to another nucleic acidmolecule, or the ability of a protein to be bound by an antibody (or tocompete with another molecule for such binding). Alternatively, such anattribute may be catalytic and thus involve the capacity of the agent tomediate a chemical reaction or response. The agents will preferably be“substantially purified.” The term “substantially purified,” as usedherein, refers to a molecule separated from substantially all othermolecules normally associated with it in its native state. Morepreferably a substantially purified molecule is the predominant speciespresent in a preparation. A substantially purified molecule may begreater than 60% free, preferably 75% free, more preferably 90% free,and most preferably 95% free from the other molecules (exclusive ofsolvent) present in the natural mixture. The term “substantiallypurified” is not intended to encompass molecules present in their nativestate.

[0157] The agents of the invention may also be recombinant. As usedherein, the term recombinant means any agent (e.g., DNA, peptide etc.),that is, or results, however indirectly, from human manipulation of anucleic acid molecule.

[0158] It is understood that the agents of the invention may be labeledwith reagents that facilitate detection of the agent (e.g., fluorescentlabels, Prober et al., Science 238:336-340 (1987); Albarella et al., EP144914; chemical labels, Sheldon et al., U.S. Pat. No. 4,582,789;Albarella et al., U.S. Pat. No. 4,563,417; modified bases, Miyoshi etal., EP 119448).

[0159] Nucleic Acid Molecules

[0160] Agents of the invention include nucleic acid molecules. In apreferred aspect of the present invention the nucleic acid moleculecomprises a nucleic acid sequence, which encodes a gamma-tocopherolmethyltransferase. As used herein a gamma-tocopherol methyltransferase(also referred to as GMT, γ-GMT, γ-MT, γ-TMT or gamma-methyltransferase)is any polypeptide that is capable of specifically catalyzing theconversion of γ-tocopherol into α-tocopherol. In certain plant speciessuch as soybean, GMT can also catalyze the conversion of δ-tocopherol toβ-tocopherol. In other plants, mainly monocotyledons such as corn andwheat, GMT can also catalyze the conversion of γ-tocotrienol toα-tocotrienol and δ-tocotrienol to β-tocotrienol. A preferredgamma-tocopherol methyltransferase is a plant or cynobacterialgamma-tocopherol methyltransferase, more preferably a gamma-tocopherolmethyltransferase that is also found in an organism selected from thegroup consisting of Arabidopsis, rice, corn, cotton, cuphea, oilseedrape, tomato, soybean, marigold, sorghum, and leek, most preferably agamma-tocopherol methyltransferase that is also found in an organismselected from the group consisting of Arabidopsis thaliana, Oryzasativa, Zea mays, Gossypium hirsutum, Cuphea pulcherrima, Brassicanapus, Lycopersicon esculentum, Glycine max, Tagetes erecta, and Liliumasiaticum. An example of a more preferred gamma-tocopherolmethyltransferase is a polypeptide with one of the amino acid sequencesset forth in SEQ ID NOs: 19-31 and 33-38.

[0161] In another embodiment of the invention, genomic DNA is used totransform any of the plants disclosed herein. Genomic DNA (e.g. SEQ IDNOs: 6 and 7) can be particularly useful for transformingmonocotyledonous plants (e.g. SEQ ID NOs: 6 and 7).

[0162] In another preferred aspect of the present invention the nucleicacid molecule of the invention comprises a nucleic acid sequenceselected from the group consisting of SEQ ID NOs: 2-17, 50, and 85, andcomplements thereof and fragments of either. In a further aspect of thepresent invention the nucleic acid molecule comprises a nucleic acidsequence encoding an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 19-31, 33, and 38 and fragments thereof.

[0163] In another preferred aspect of the present invention the nucleicacid molecule comprises a nucleic acid sequence, which encodes a2-methyl-6-phytylplastoquinol/2-methyl-6-solanylplastoquinol-9methyltransferase. As used herein a2-methyl-6-phytylplastoquinol/2-methyl-6-solanylplastoquinol-9methyltransferase (MT1) is any protein that is capable of specificallycatalyzing the conversion of 2-methyl-6-phytylplastoquinol,2-methyl-5-phytylplastoquinol or 2-methyl-3-phytylplastoquinol to2,3-dimethyl-6-phytylplastoquinol. A preferred MT 1 is a cyanobacterialMT 1, more preferably an MT 1 that is also found in an organism selectedfrom the group consisting of Anabaena, Synechococcus and Prochlorococcusmarinus. An example of a more preferred MT 1 is a polypeptide with theamino acid sequence selected from the group consisting of SEQ ID NOs:46-49.

[0164] In another preferred aspect of the present invention the nucleicacid molecule of the invention comprises a nucleic acid sequenceselected from the group consisting of SEQ ID NOs: 42-45 and complementsthereof and fragments of either. In a further aspect of the presentinvention the nucleic acid molecule comprises a nucleic acid sequenceencoding an amino acid sequence selected from the group consisting ofSEQ ID NOs: 46-49 and fragments thereof.

[0165] In another preferred aspect of the present invention a nucleicacid molecule comprises nucleotide sequences encoding a plastid transitpeptide operably fused to a nucleic acid molecule that encodes a proteinor fragment of the present invention.

[0166] It is understood that in a further aspect of the presentinvention, the nucleic acids can encode a protein that differs from anyof the proteins in that one or more amino acids have been deleted,substituted or added without altering the function. For example, it isunderstood that codons capable of coding for such conservative aminoacid substitutions are known in the art.

[0167] One subset of the nucleic acid molecules of the invention isfragment nucleic acids molecules. Fragment nucleic acid molecules mayconsist of significant portion(s) of, or indeed most of, the nucleicacid molecules of the invention, such as those specifically disclosed.Alternatively, the fragments may comprise smaller oligonucleotides(having from about 15 to about 400 nucleotide residues and morepreferably, about 15 to about 30 nucleotide residues, or about 50 toabout 100 nucleotide residues, or about 100 to about 200 nucleotideresidues, or about 200 to about 400 nucleotide residues, or about 275 toabout 350 nucleotide residues).

[0168] A fragment of one or more of the nucleic acid molecules of theinvention may be a probe and specifically a PCR probe. A PCR probe is anucleic acid molecule capable of initiating a polymerase activity whilein a double-stranded structure with another nucleic acid. Variousmethods for determining the structure of PCR probes and PCR techniquesexist in the art. Computer generated searches using programs such asPrimer3 (www.genome.wi.mit.edu/cgi-bin/primer/primer3.cgi), STSPipeline(www.genome.wi.mit.edu/cgi-bin/www-STS_Pipeline), or GeneUp (Pesole etal., BioTechniques 25:112-123 (1998)), for example, can be used toidentify potential PCR primers.

[0169] Another subset of the nucleic acid molecules of the inventioninclude nucleic acid molecules that encode a polypeptide or fragmentthereof.

[0170] Nucleic acid molecules or fragments thereof of the presentinvention are capable of specifically hybridizing to other nucleic acidmolecules under certain circumstances. Nucleic acid molecules of thepresent invention include those that specifically hybridize to nucleicacid molecules having a nucleic acid sequence selected from the groupconsisting of SEQ ID NOs: 2-17, 50, and 85, and complements thereof.Nucleic acid molecules of the present invention also include those thatspecifically hybridize to nucleic acid molecules encoding an amino acidsequence selected from SEQ ID NOs: 19-31 and 33-38 and fragmentsthereof.

[0171] As used herein, two nucleic acid molecules are said to be capableof specifically hybridizing to one another if the two molecules arecapable of forming an anti-parallel, double-stranded nucleic acidstructure.

[0172] A nucleic acid molecule is said to be the “complement” of anothernucleic acid molecule if they exhibit complete complementarity. As usedherein, molecules are said to exhibit “complete complementarity” whenevery nucleotide of one of the molecules is complementary to anucleotide of the other. Two molecules are said to be “minimallycomplementary” if they can hybridize to one another with sufficientstability to permit them to remain annealed to one another under atleast conventional “low-stringency” conditions. Similarly, the moleculesare said to be “complementary” if they can hybridize to one another withsufficient stability to permit them to remain annealed to one anotherunder conventional “high-stringency” conditions. Conventional stringencyconditions are described by Sambrook et al., Molecular Cloning, ALaboratory Manual, 2nd Ed., Cold Spring Harbor Press, Cold SpringHarbor, New York (1989), and by Haymes et al., Nucleic AcidHybridization, A Practical Approach, IRL Press, Washington, D.C. (1985).Departures from complete complementarity are therefore permissible, aslong as such departures do not completely preclude the capacity of themolecules to form a double-stranded structure. Thus, in order for anucleic acid molecule to serve as a primer or probe it need only besufficiently complementary in sequence to be able to form a stabledouble-stranded structure under the particular solvent and saltconcentrations employed.

[0173] Appropriate stringency conditions which promote DNA hybridizationare, for example, 6.0×sodium chloride/sodium citrate (SSC) at about 45°C., followed by a wash of 2.0×SSC at 20-25° C., are known to thoseskilled in the art or can be found in Current Protocols in MolecularBiology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. For example, thesalt concentration in the wash step can be selected from a lowstringency of about 2.0×SSC at 50° C. to a high stringency of about 0.2X SSC at 65° C. In addition, the temperature in the wash step can beincreased from low stringency conditions at room temperature, about 22°C., to high stringency conditions at about 65° C. Both temperature andsalt may be varied, or either the temperature or the salt concentrationmay be held constant while the other variable is changed.

[0174] In a preferred embodiment, a nucleic acid of the presentinvention will specifically hybridize to one or more of the nucleic acidmolecules set forth in SEQ ID NOs: 2-17, 50, and 85 and complementsthereof under moderately stringent conditions, for example at about2.0×SSC and about 65° C.

[0175] In a particularly preferred embodiment, a nucleic acid of thepresent invention will include those nucleic acid molecules thatspecifically hybridize to one or more of the nucleic acid molecules setforth in SEQ ID NOs: 2-17, 50, and 85 and complements thereof under highstringency conditions such as 0.2×SSC and about 65° C.

[0176] In one aspect of the present invention, the nucleic acidmolecules of the present invention have one or more of the nucleic acidsequences set forth in SEQ ID NOs: 2-17, 50, and 85 and complementsthereof. In another aspect of the present invention, one or more of thenucleic acid molecules of the present invention share between 100% and90% sequence identity with one or more of the nucleic acid sequences setforth in SEQ ID NOs: 2-17, 50, and 85 and complements thereof andfragments of either. In a further aspect of the present invention, oneor more of the nucleic acid molecules of the present invention sharebetween 100% and 95% sequence identity with one or more of the nucleicacid sequences set forth in SEQ ID NOs: 2-17, 50, and 85, complementsthereof, and fragments of either. In a more preferred aspect of thepresent invention, one or more of the nucleic acid molecules of thepresent invention share between 100% and 98% sequence identity with oneor more of the nucleic acid sequences set forth in SEQ ID NOs: 2-17, 50,and 85, complements thereof and fragments of either. In an even morepreferred aspect of the present invention, one or more of the nucleicacid molecules of the present invention share between 100% and 99%sequence identity with one or more of the sequences set forth in SEQ IDNOs: 2-17, 50, and 85, complements thereof, and fragments of either.

[0177] In a preferred embodiment the percent identity calculations areperformed using BLASTN or BLASTP (default, parameters, version 2.0.8,Altschul et al., Nucleic Acids Res. 25: 3389-3402 (1997)).

[0178] A nucleic acid molecule of the invention can also encode ahomolog polypeptide. As used herein, a homolog polypeptide molecule orfragment thereof is a counterpart protein molecule or fragment thereofin a second species (e.g., corn rubisco small subunit is a homolog ofArabidopsis rubisco small subunit). A homolog can also be generated bymolecular evolution or DNA shuffling techniques, so that the moleculeretains at least one functional or structure characteristic of theoriginal polypeptide (see, for example, U.S. Pat. No. 5,811,238).

[0179] In another embodiment, the homolog is selected from the groupconsisting of alfalfa, Arabidopsis, barley, Brassica campestris, oilseedrape, broccoli, cabbage, canola, citrus, cotton, garlic, oat, onion,flax, an ornamental plant, peanut, pepper, potato, rapeseed, rice, rye,sorghum, strawberry, sugarcane, sugarbeet, tomato, wheat, poplar, pine,fir, eucalyptus, apple, lettuce, lentils, grape, banana, tea, turfgrasses, sunflower, soybean, corn, Phaseolus, crambe, mustard, castorbean, sesame, cottonseed, linseed, safflower, and oil palm. Moreparticularly, preferred homologs are selected from canola, corn,Brassica campestris, oilseed rape, soybean, crambe, mustard, castorbean, peanut, sesame, cottonseed, linseed, rapeseed, safflower, oilpalm, flax, and sunflower. In an even more preferred embodiment, thehomolog is selected from the group consisting of canola, rapeseed, corn,Brassica campestris, Brassica napus, soybean, sunflower, safflower, oilpalms, and peanut. In a particularly preferred embodiment, the homologis soybean. In a particularly preferred embodiment, the homolog iscanola. In a particularly preferred embodiment, the homolog is Brassicanapus.

[0180] In another further aspect of the present invention, nucleic acidmolecules of the present invention can comprise sequences that differfrom those encoding a polypeptide or fragment thereof in SEQ ID NOs:19-31 and 33-38 due to the fact that a polypeptide can have one or moreconservative amino acid changes, and nucleic acid sequences coding forthe polypeptide can therefore have sequence differences. It isunderstood that codons capable of coding for such conservative aminoacid substitutions are known in the art.

[0181] It is well known in the art that one or more amino acids in anative sequence can be substituted with other amino acid(s), the chargeand polarity of which are similar to that of the native amino acid,i.e., a conservative amino acid substitution. Conservative substitutesfor an amino acid within the native polypeptide sequence can be selectedfrom other members of the class to which the amino acid belongs. Aminoacids can be divided into the following four groups: (1) acidic aminoacids, (2) basic amino acids, (3) neutral polar amino acids, and (4)neutral, nonpolar amino acids. Representative amino acids within thesevarious groups include, but are not limited to, (1) acidic (negativelycharged) amino acids such as aspartic acid and glutamic acid; (2) basic(positively charged) amino acids such as arginine, histidine, andlysine; (3) neutral polar amino acids such as glycine, serine,threonine, cysteine, cystine, tyrosine, asparagine, and glutamine; and(4) neutral nonpolar (hydrophobic) amino acids such as alanine, leucine,isoleucine, valine, proline, phenylalanine, tryptophan, and methionine.

[0182] Conservative amino acid substitution within the nativepolypeptide sequence can be made by replacing one amino acid from withinone of these groups with another amino acid from within the same group.In a preferred aspect, biologically functional equivalents of theproteins or fragments thereof of the present invention can have ten orfewer conservative amino acid changes, more preferably seven or fewerconservative amino acid changes, and most preferably five or fewerconservative amino acid changes. The encoding nucleotide sequence willthus have corresponding base substitutions, permitting it to encodebiologically functional equivalent forms of the polypeptides of thepresent invention.

[0183] It is understood that certain amino acids may be substituted forother amino acids in a protein structure without appreciable loss ofinteractive binding capacity with structures such as, for example,antigen-binding regions of antibodies or binding sites on substratemolecules. Because it is the interactive capacity and nature of aprotein that defines that protein's biological functional activity,certain amino acid sequence substitutions can be made in a proteinsequence and, of course, its underlying DNA coding sequence and,nevertheless, a protein with like properties can still be obtained. Itis thus contemplated by the inventors that various changes may be madein the peptide sequences of the proteins or fragments of the presentinvention, or corresponding DNA sequences that encode said peptides,without appreciable loss of their biological utility or activity. It isunderstood that codons capable of coding for such amino acid changes areknown in the art.

[0184] In making such changes, the hydropathic index of amino acids maybe considered. The importance of the hydropathic amino acid index inconferring interactive biological function on a protein is generallyunderstood in the art (Kyte and Doolittle, J. Mol. Biol. 157, 105-132(1982)). It is accepted that the relative hydropathic character of theamino acid contributes to the secondary structure of the resultantpolypeptide, which in turn defines the interaction of the protein withother molecules, for example, enzymes, substrates, receptors, DNA,antibodies, antigens, and the like.

[0185] Each amino acid has been assigned a hydropathic index on thebasis of its hydrophobicity and charge characteristics (Kyte andDoolittle, J. Mol. Biol. 157:105-132 (1982)); these are isoleucine(+4.5), valine (+4.2), leucine (+3.8), phenylalanine (+2.8),cysteine/cystine (+2.5), methionine (+1.9), alanine (+1.8), glycine(−0.4), threonine (−0.7), serine (−0.8), tryptophan (−0.9), tyrosine(−1.3), proline (−1.6), histidine (−3.2), glutamate (−3.5), glutamine(−3.5), aspartate (−3.5), asparagine (−3.5), lysine (−3.9), and arginine(−4.5).

[0186] In making such changes, the substitution of amino acids whosehydropathic indices are within ±2 is preferred, those that are within ±1are particularly preferred, and those within ±0.5 are even moreparticularly preferred.

[0187] It is also understood in the art that the substitution of likeamino acids can be made effectively on the basis of hydrophilicity. U.S.Pat. No. 4,554,101 states that the greatest local average hydrophilicityof a protein, as governed by the hydrophilicity of its adjacent aminoacids, correlates with a biological property of the protein.

[0188] As detailed in U.S. Pat. No. 4,554,101, the followinghydrophilicity values have been assigned to amino acid residues:arginine (+3.0), lysine (+3.0), aspartate (+3.0±1), glutamate (+3.0±1),serine (+0.3), asparagine (+0.2), glutamine (+0.2), glycine (0),threonine (−0.4), proline (−0.5±1), alanine (−0.5), histidine (−0.5),cysteine (−1.0), methionine (−1.3), valine (−1.5), leucine (−1.8),isoleucine (−1.8), tyrosine (−2.3), phenylalanine (−2.5), and tryptophan(−3.4).

[0189] In making such changes, the substitution of amino acids whosehydrophilicity values are within ±2 is preferred, those that are within±1 are particularly preferred, and those within ±0.5 are even moreparticularly preferred.

[0190] In a further aspect of the present invention, one or more of thenucleic acid molecules of the present invention differ in nucleic acidsequence from those for which a specific sequence is provided hereinbecause one or more codons has been replaced with a codon that encodes aconservative substitution of the amino acid originally encoded.

[0191] Agents of the invention include nucleic acid molecules thatencode at least about a contiguous 10 amino acid region of a polypeptideof the present invention, more preferably at least about a contiguous25, 40, 50, 100, or 125 amino acid region of a polypeptide of thepresent invention.

[0192] In a preferred embodiment, any of the nucleic acid molecules ofthe present invention can be operably linked to a promoter region thatfunctions in a plant cell to cause the production of an mRNA molecule,where the nucleic acid molecule that is linked to the promoter isheterologous with respect to that promoter. As used herein,“heterologous” means not naturally occurring together.

[0193] Protein and Peptide Molecules

[0194] A class of agents includes one or more of the polypeptidemolecules encoded by a nucleic acid agent of the invention. A particularpreferred class of proteins is that having an amino acid sequenceselected from the group consisting of SEQ ID NOs: 19-31 and 33-38 andfragments thereof. Polypeptide agents may have C-terminal or N-terminalamino acid sequence extensions. One class of N-terminal extensionsemployed in a preferred embodiment are plastid transit peptides. Whenemployed, plastid transit peptides can be operatively linked to theN-terminal sequence, thereby permitting the localization of the agentpolypeptides to plastids. In a preferred embodiment the plastidtargeting sequence is a CTP1 sequence (SEQ ID NO: 32). See WO 00/61771.

[0195] In a preferred aspect a protein of the present invention istargeted to a plastid using either a native transit peptide sequence ora heterologous transit peptide sequence. In the case of nucleic acidsequences corresponding to nucleic acid sequences of non-higher plantorganisms such as cyanobacteria, such nucleic acid sequences can bemodified to attach the coding sequence of the protein to a nucleic acidsequence of a plastid targeting peptide. Examples of cynobacterialnucleic acid sequences that can be so attached are those having aminoacid sequence set forth in SEQ ID NOs: 42-45.

[0196] As used herein, the term “protein,” “peptide molecule,” or“polypeptide” includes any molecule that comprises five or more aminoacids. It is well known in the art that protein, peptide or polypeptidemolecules may undergo modification, including post-translationalmodifications, such as, but not limited to, disulfide bond formation,glycosylation, phosphorylation, or oligomerization. Thus, as usedherein, the term “protein,” “peptide molecule,” or “polypeptide”includes any protein that is modified by any biological ornon-biological process. The terms “amino acid” and “amino acids” referto all naturally occurring L-amino acids. This definition is meant toinclude norleucine, norvaline, omithine, homocysteine, and homoserine.

[0197] One or more of the protein or fragments thereof, peptidemolecules, or polypeptide molecules may be produced via chemicalsynthesis, or more preferably, by expression in a suitable bacterial oreukaryotic host. Suitable methods for expression are described bySambrook et al., In: Molecular Cloning, A Laboratory Manual, 2ndEdition, Cold Spring Harbor Press, Cold Spring Harbor, New York (1989)or similar texts.

[0198] A “protein fragment” is a peptide or polypeptide molecule whoseamino acid sequence comprises a subset of the amino acid sequence ofthat protein. A protein or fragment thereof that comprises one or moreadditional peptide regions not derived from that protein is a “fusion”protein. Such molecules may be derivatized to contain carbohydrate orother moieties (such as keyhole limpet hemocyanin). Fusion protein orpeptide molecules of the invention are preferably produced viarecombinant means.

[0199] Another class of agents comprise protein, peptide molecules, orpolypeptide molecules or fragments or fusions thereof comprising SEQ IDNOs: 19-31 and 33-38 and fragments thereof in which conservative,non-essential or non-relevant amino acid residues have been added,replaced or deleted. Computerized means for designing modifications inprotein structure are known in the art (Dahiyat and Mayo, Science278:82-87 (1997)).

[0200] A protein, peptide or polypeptide of the invention can also be ahomolog protein, peptide or polypeptide. As used herein, a homologprotein, peptide or polypeptide or fragment thereof is a counterpartprotein, peptide or polypeptide or fragment thereof in a second species.A homolog can also be generated by molecular evolution or DNA shufflingtechniques, so that the molecule retains at least one functional orstructure characteristic of the original (see, for example, U.S. Pat.No. 5,811,238).

[0201] In another embodiment, the homolog is selected from the groupconsisting of alfalfa, Arabidopsis, barley, broccoli, cabbage, canola,citrus, cotton, garlic, oat, onion, flax, an ornamental plant, peanut,pepper, potato, rapeseed, rice, rye, sorghum, strawberry, sugarcane,sugarbeet, tomato, wheat, poplar, pine, fir, eucalyptus, apple, lettuce,lentils, grape, banana, tea, turf grasses, sunflower, soybean, corn, andPhaseolus. More particularly, preferred homologs are selected fromcanola, rapeseed, corn, Brassica campestris, oilseed rape, soybean,crambe, mustard, castor bean, peanut, sesame, cottonseed, linseed,safflower, oil palm, flax, and sunflower. In an even more preferredembodiment, the homolog is selected from the group consisting of canola,rapeseed, corn, Brassica campestris, oilseed rape, soybean, sunflower,safflower, oil palms, and peanut. In a preferred embodiment, the homologis soybean. In a preferred embodiment, the homolog is canola. In apreferred embodiment, the homolog is Brassica napus.

[0202] In a preferred embodiment, the nucleic acid molecules of thepresent invention or complements and fragments of either can be utilizedto obtain such homologs.

[0203] Agents of the invention include proteins and fragments thereofcomprising at least about a contiguous 10 amino acid region preferablycomprising at least about a contiguous 20 amino acid region, even morepreferably comprising at least about a contiguous 25, 35, 50, 75 or 100amino acid region of a protein of the present invention. In anotherpreferred embodiment, the proteins of the present invention includebetween about 10 and about 25 contiguous amino acid region, morepreferably between about 20 and about 50 contiguous amino acid region,and even more preferably between about 40 and about 80 contiguous aminoacid region.

[0204] Plant Constructs and Plant Transformants

[0205] One or more of the nucleic acid molecules of the invention may beused in plant transformation or transfection. Exogenous genetic materialmay be transferred into a plant cell and the plant cell regenerated intoa whole, fertile or sterile plant. Exogenous genetic material is anygenetic material, whether naturally occurring or otherwise, from anysource that is capable of being inserted into any organism.

[0206] In a preferred aspect of the present invention the exogenousgenetic material comprises a nucleic acid sequence that encodes agamma-tocopherol methyltransferase. In a particularly preferredembodiment of the present invention, the exogenous genetic material ofthe invention comprises a nucleic acid sequence of SEQ ID NO: 2. In afurther aspect of the present invention the exogenous genetic materialcomprises a nucleic acid sequence encoding an amino acid sequenceselected from the group consisting of SEQ ID NOs: 19-31, 33-38, 39-41,and 46-49 and fragments thereof.

[0207] In another preferred aspect of the present invention theexogenous genetic material comprises a nucleic acid sequence thatencodes a 2-methyl-6-phytylplastoquinol/2-methyl-6-solanylplastoquinol-9methyltransferase. In another preferred aspect of the present inventionthe exogenous genetic material of the invention comprises a nucleic acidsequence selected from the group consisting of SEQ ID NOs: 42-45 andcomplements thereof and fragments of either. In a further aspect of thepresent invention the exogenous genetic material comprises a nucleicacid sequence encoding an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 46-49 and fragments thereof. In a furtheraspect of the present invention, the nucleic acid sequences of theinvention also encode peptides involved in intracellular localization,export, or post-translational modification.

[0208] In an embodiment of the present invention, exogenous geneticmaterial comprising a GMT or fragment thereof is introduced into a plantwith one or more additional genes. In another embodiment of the presentinvention, exogenous genetic material comprising a MT1 or fragmentthereof is introduced into a plant with one or more additional genes. Inone embodiment, preferred combinations of genes include two or more ofthe following genes: tyrA, slr1736, ATPT2, dxs, dxr, GGPPS, HPPD, GMT,MT1, tMT2, AANT1, slr 1737, or a plant ortholog and an antisenseconstruct for homogentisic acid dioxygenase (Kridl et al., Seed Sci.Res. 1:209:219 (1991); Keegstra, Cell 56(2):247-53 (1989); Nawrath, etal., Proc. Natl. Acad. Sci. U.S.A. 91:12760-12764 (1994); Xia et al., J.Gen. Microbiol. 138:1309-1316 (1992); Cyanobase,www.kazusa.or.jp/cyanobase; Lois et al., Proc. Natl. Acad. Sci. U.S.A.95 (5):2105-2110 (1998); Takahashi et al. Proc. Natl. Acad. Sci. U.S.A.95 (17), 9879-9884 (1998); Norris et al., Plant Physiol. 117:1317-1323(1998); Bartley and Scolnik, Plant Physiol. 104:1469-1470 (1994), Smithet al., Plant J. 11: 83-92 (1997); WO 00/32757; WO 00/10380; SaintGuily, et al., Plant Physiol., 100(2):1069-1071 (1992); Sato et al., J.DNA Res. 7 (1):31-63 (2000)). In such combinations, one or more of thegene products can be directed to the plastid by the use of a plastidtargeting sequence. Alternatively, one or more of the gene products canbe localized in the cytoplasm. In a preferred aspect the gene productsof tyrA and HPPD are targeted to the cytoplasm. Such genes can beintroduced, for example, with the MT1 or GMT or both or fragment ofeither or both on a single construct, introduced on different constructsbut the same transformation event or introduced into separate plantsfollowed by one or more crosses to generate the desired combination ofgenes. In such combinations, a preferred promoter is a napin promoterand a preferred plastid targeting sequence is a CTP1 sequence. It ispreferred that gene products are targeted to the plastid.

[0209] A particularly preferred combination that can be introduced is anucleic acid molecule encoding a GMT polypeptide and a nucleic acidmolecule encoding an MT1 polypeptide, where both polypeptides aretargeted to the plastid and where one of such polypeptides is presentand the other is introduced. Both nucleic acid sequences encoding suchpolypeptides are introduced using a single construct, or eachpolypeptide is introduced on separate constructs.

[0210] Another particularly preferred combination that can be introducedis a nucleic acid molecule encoding an MT1 protein and a nucleic acidmolecule that results in the down regulation of a GMT protein. In suchan aspect, it is preferred that the plant accumulates eitherγ-tocopherol or γ-tocotrienol or both.

[0211] Such genetic material may be transferred into eithermonocotyledons or dicotyledons including, but not limited to canola,corn, soybean, Arabidopsis phaseolus, peanut, alfalfa, wheat, rice, oat,sorghum, rapeseed, rye, tritordeum, millet, fescue, perennial rye-grass,sugarcane, cranberry, papaya, banana, safflower, oil palms, flax,muskmelon, apple, cucumber, dendrobium, gladiolus, chrysanthemum,liliacea, cotton, eucalyptus, sunflower, Brassica campestris, Brassicanapus, turfgrass, sugarbeet, coffee and dioscorea (Christou, In:Particle Bombardment for Genetic Engineering of Plants, BiotechnologyIntelligence Unit. Academic Press, San Diego, Calif. (1996)), withcanola, corn, Brassica campestris, Brassica napus, rapeseed, soybean,crambe, mustard, castor bean, peanut, sesame, cottonseed, linseed,safflower, oil palm, flax, and sunflower preferred, and canola,rapeseed, corn, Brassica campestris, Brassica napus, soybean, sunflower,safflower, oil palms, and peanut preferred. In a more preferredembodiment, the genetic material is transferred into canola. In anothermore preferred embodiment, the genetic material is transferred intoBrassica napus. In another particularly preferred embodiment, thegenetic material is transferred into soybean. In another particularlypreferred embodiment of the present invention, the genetic material istransferred into soybean line 3244.

[0212] Transfer of a nucleic acid molecule that encodes a protein canresult in expression or overexpression of that polypeptide in atransformed cell or transgenic plant. One or more of the proteins orfragments thereof encoded by nucleic acid molecules of the invention maybe overexpressed in a transformed cell or transformed plant. Suchexpression or overexpression may be the result of transient or stabletransfer of the exogenous genetic material.

[0213] In a preferred embodiment, expression or overexpression of apolypeptide of the present invention in a plant provides in that plant,relative to an untransformed plant with a similar genetic background, anincreased level of tocopherols.

[0214] In a preferred embodiment, expression, or overexpression of apolypeptide of the present invention in a plant provides in that plant,relative to an untransformed plant with a similar genetic background, anincreased level of α-tocopherols.

[0215] In a preferred embodiment, expression, or overexpression of apolypeptide of the present invention in a plant provides in that plant,relative to an untransformed plant with a similar genetic background, anincreased level of γ-tocopherols.

[0216] In a preferred embodiment, reduction of the expression,expression, or overexpression of a polypeptide of the present inventionin a plant provides in that plant, relative to an untransformed plantwith a similar genetic background, an increased level of δ-tocopherols.

[0217] In a preferred embodiment, reduction of the expression,expression or overexpression of a polypeptide of the present inventionin a plant provides in that plant, relative to an untransformed plantwith a similar genetic background, an increased level of tocotrienols.

[0218] In a preferred embodiment, reduction of the expression,expression, or overexpression of a polypeptide of the present inventionin a plant provides in that plant, relative to an untransformed plantwith a similar genetic background, an increased level of α-tocotrienols.

[0219] In a preferred embodiment, reduction of the expression,expression, or overexpression of a polypeptide of the present inventionin a plant provides in that plant, relative to an untransformed plantwith a similar genetic background, an increased level of γ-tocotrienols.

[0220] In a preferred embodiment, reduction of the expression,expression, or overexpression of a polypeptide of the present inventionin a plant provides in that plant, relative to an untransformed plantwith a similar genetic background, an increased level of δ-tocotrienols.

[0221] In another embodiment, reduction of the expression, expression,overexpression of a polypeptide of the present invention in a plantprovides in that plant, or a tissue of that plant, relative to anuntransformed plant or plant tissue, with a similar genetic background,an increased level of an MT1 or GMT protein or both or fragment ofeither.

[0222] In some embodiments, the levels of one or more products of thetocopherol biosynthesis pathway, including any one or more oftocopherols, α-tocopherols, γ-tocopherols, δ-tocopherols, β-tocopherols,tocotrienols, α-tocotrienols, γ-tocotrienols, δ-tocotrienols,β-tocotrienols, are increased by greater than about 10%, or morepreferably greater than about 25%, 50%, 200%, 1,000%, 2,000%, 2,500% or25,000%. The levels of products may be increased throughout an organismsuch as a plant or localized in one or more specific organs or tissuesof the organism. For example the levels of products may be increased inone or more of the tissues and organs of a plant including withoutlimitation: roots, tubers, stems, leaves, stalks, fruit, berries, nuts,bark, pods, seeds and flowers. A preferred organ is a seed.

[0223] In some embodiments, the levels of tocopherols or a species suchas α-tocopherol may be altered. In some embodiments, the levels oftocotrienols may be altered. Such alteration can be compared to a plantwith a similar background.

[0224] In another embodiment, either the α-tocopherol level,α-tocotrienol level, or both of plants that natively produce high levelsof either α-tocopherol, α-tocotrienol or both (e.g., sunflowers), can beincreased by the introduction of a gene coding for an MT1 enzyme.

[0225] In a preferred aspect, a similar genetic background is abackground where the organisms being compared share about 50% or greaterof their nuclear genetic material. In a more preferred aspect a similargenetic background is a background where the organisms being comparedshare about 75% or greater, even more preferably about 90% or greater oftheir nuclear genetic material. In another even more preferable aspect,a similar genetic background is a background where the organisms beingcompared are plants, and the plants are isogenic except for any geneticmaterial originally introduced using plant transformation techniques.

[0226] In another preferred embodiment, reduction of the expression,expression, overexpression of a polypeptide of the present invention ina transformed plant may provide tolerance to a variety of stress, e.g.oxidative stress tolerance such as to oxygen or ozone, UV tolerance,cold tolerance, or fungal/microbial pathogen tolerance.

[0227] As used herein in a preferred aspect, a tolerance or resistanceto stress is determined by the ability of a plant, when challenged by astress such as cold to produce a plant having a higher yield than onewithout such tolerance or resistance to stress. In a particularlypreferred aspect of the present invention, the tolerance or resistanceto stress is measured relative to a plant with a similar geneticbackground to the tolerant or resistance plant except that the plantreduces the expression, expresses or over expresses a protein orfragment thereof of the present invention.

[0228] Exogenous genetic material may be transferred into a host cell bythe use of a DNA vector or construct designed for such a purpose. Designof such a vector is generally within the skill of the art (See, PlantMolecular Biology: A Laboratory Manual, Clark (ed.), Springer, N.Y.(1997)).

[0229] A construct or vector may include a plant promoter to express thepolypeptide of choice. In a preferred embodiment, any nucleic acidmolecules described herein can be operably linked to a promoter regionwhich functions in a plant cell to cause the production of an mRNAmolecule. For example, any promoter that functions in a plant cell tocause the production of an mRNA molecule, such as those promotersdescribed herein, without limitation, can be used. In a preferredembodiment, the promoter is a plant promoter.

[0230] A number of promoters that are active in plant cells have beendescribed in the literature. These include the nopaline synthase (NOS)promoter (Ebert et al., Proc. Natl. Acad. Sci. (U.S.A.) 84:5745-5749(1987)), the octopine synthase (OCS) promoter (which is carried ontumor-inducing plasmids of Agrobacterium tumefaciens), the caulimoviruspromoters such as the cauliflower mosaic virus (CaMV) 19S promoter(Lawton et al., Plant Mol. Biol. 9:315-324 (1987)) and the CaMV 35Spromoter (Odell et al., Nature 313:810-812 (1985)), the figwort mosaicvirus 35S-promoter, the light-inducible promoter from the small subunitof ribulose-1,5-bis-phosphate carboxylase (ssRUBISCO), the Adh promoter(Walker et al., Proc. Natl. Acad. Sci. (U.S.A.) 84:6624-6628 (1987)),the sucrose synthase promoter (Yang et al., Proc. Natl. Acad. Sci.(U.S.A.) 87:4144-4148 (1990)), the R gene complex promoter (Chandler etal., The Plant Cell 1:1175-1183 (1989)) and the chlorophyll a/b bindingprotein gene promoter, etc. These promoters have been used to create DNAconstructs that have been expressed in plants; see, e.g., PCTpublication WO 84/02913. The CaMV 35S promoters are preferred for use inplants. Promoters known or found to cause transcription of DNA in plantcells can be used in the invention.

[0231] For the purpose of expression in source tissues of the plant,such as the leaf, seed, root or stem, it is preferred that the promotersutilized have relatively high expression in these specific tissues.Tissue-specific expression of a protein of the present invention is aparticularly preferred embodiment. For this purpose, one may choose froma number of promoters for genes with tissue- or cell-specific orenhanced expression. Examples of such promoters reported in theliterature include the chloroplast glutamine synthetase GS2 promoterfrom pea (Edwards et al., Proc. Natl. Acad. Sci. (U.S.A.) 87:3459-3463(1990)), the chloroplast fructose-1,6-biphosphatase (FBPase) promoterfrom wheat (Lloyd et al., Mol. Gen. Genet, 225:209-216 (1991)), thenuclear photosynthetic ST-LS1 promoter from potato (Stockhaus et al.,EMBO J. 8:2445-2451 (1989)), the serine/threonine kinase (PAL) promoterand the glucoamylase (CHS) promoter from Arabidopsis thaliana. Alsoreported to be active in photosynthetically active tissues are theribulose-1,5-bisphosphate carboxylase (RbcS) promoter from eastern larch(Larix laricina), the promoter for the cab gene, cab6, from pine(Yamamoto et al., Plant Cell Physiol. 35:773-778 (1994)), the promoterfor the Cab-1 gene from wheat (Fejes et al., Plant Mol. Biol. 15:921-932(1990)), the promoter for the CAB-1 gene from spinach (Lubberstedt etal., Plant Physiol. 104:997-1006 (1994)), the promoter for the cab1Rgene from rice (Luan et al., Plant Cell. 4:971-981 (1992)), thepyruvate, orthophosphate dikinase (PPDK) promoter from corn (Matsuoka etal., Proc. Natl. Acad. Sci. (U.S.A.) 90: 9586-9590 (1993)), the promoterfor the tobacco Lhcb1*2 gene (Cerdan et al., Plant Mol. Biol. 33:245-255(1997)), the Arabidopsis thaliana SUC2 sucrose-H+ symporter promoter(Truernit et al., Planta. 196:564-570 (1995)) and the promoter for thethylakoid membrane proteins from spinach (psaD, psaF, psaE, PC, FNR,atpC, atpD, cab, rbcS). Other promoters for the chlorophyll a/b-bindingproteins may also be utilized in the invention, such as the promotersfor LhcB gene and PsbP gene from white mustard (Sinapis alba; Kretsch etal., Plant Mol. Biol. 28:219-229 (1995)).

[0232] For the purpose of expression in sink tissues of the plant, suchas the tuber of the potato plant, the fruit of tomato, or the seed ofcorn, wheat, rice and barley, it is preferred that the promotersutilized in the invention have relatively high expression in thesespecific tissues. A number of promoters for genes with tuber-specific ortuber-enhanced expression are known, including the class I patatinpromoter (Bevan et al., EMBO J. 8:1899-1906 (1986); Jefferson et al.,Plant Mol. Biol. 14:995-1006 (1990)), the promoter for the potato tuberADPGPP genes, both the large and small subunits, the sucrose synthasepromoter (Salanoubat and Belliard, Gene 60:47-56 (1987), Salanoubat andBelliard, Gene 84:181-185 (1989)), the promoter for the major tuberproteins including the 22 kd protein complexes and protease inhibitors(Hannapel, Plant Physiol. 101:703-704 (1993)), the promoter for thegranule-bound starch synthase gene (GBSS) (Visser et al., Plant Mol.Biol. 17:691-699 (1991)) and other class I and II patatins promoters(Koster-Topfer et al., Mol. Gen. Genet. 219:390-396 (1989); Mignery etal., Gene. 62:27-44 (1988)).

[0233] Other promoters can also be used to express a polypeptide inspecific tissues, such as seeds or fruits. Indeed, in a preferredembodiment, the promoter used is a seed specific promoter. Examples ofsuch promoters include the 5′ regulatory regions from such genes asnapin (Kridl et al., Seed Sci. Res. 1:209:219 (1991)), phaseolin(Bustos, et al., Plant Cell, 1(9):839-853 (1989)), soybean trypsininhibitor (Riggs, et al., Plant Cell 1(6):609-621 (1989)), ACP (Baerson,et al., Plant Mol. Biol., 22(2):255-267 (1993)) stearoyl-ACP desaturase(Slocombe, et al., Plant Physiol. 104(4):167-176 (1994)), soybean α′subunit of β-conglycinin (soy 7s, (Chen et al., Proc. Natl. Acad. Sci.,83:8560-8564 (1986))), and oleosin (see, for example, Hong, et al.,Plant Mol. Biol, 34(3):549-555 (1997)). Further examples include thepromoter for β-conglycinin (Chen et al., Dev. Genet. 10: 112-122(1989)). Also included are the zeins, which are a group of storageproteins found in corn endosperm. Genomic clones for zein genes havebeen isolated (Pedersen et al., Cell 29:1015-1026 (1982), and Russell etal., Transgenic Res. 6(2):157-168) and the promoters from these clones,including the 15 kD, 16 kD, 19 kD, 22 kD, 27 kD and genes, could also beused. Other promoters known to function, for example, in corn includethe promoters for the following genes: waxy, Brittle, Shrunken 2,Branching enzymes I and II, starch synthases, debranching enzymes,oleosins, glutelins and sucrose synthases. A particularly preferredpromoter for corn endosperm expression is the promoter for the glutelingene from rice, more particularly the Osgt-1 promoter (Zheng et al.,Mol. Cell Biol. 13:5829-5842 (1993)). Examples of promoters suitable forexpression in wheat include those promoters for the ADPglucosepyrosynthase (ADPGPP) subunits, the granule bound and other starchsynthase, the branching and debranching enzymes, theembryogenesis-abundant proteins, the gliadins and the glutenins.Examples of such promoters in rice include those promoters for theADPGPP subunits, the granule bound and other starch synthase, thebranching enzymes, the debranching enzymes, sucrose synthases and theglutelins. A particularly preferred promoter is the promoter for riceglutelin, Osgt-1. Examples of such promoters for barley include thosefor the ADPGPP subunits, the granule bound and other starch synthase,the branching enzymes, the debranching enzymes, sucrose synthases, thehordeins, the embryo globulins and the aleurone specific proteins. Apreferred promoter for expression in the seed is a napin promoter.Another preferred promoter for expression is an Arcelin 5 promoter.

[0234] Root specific promoters may also be used. An example of such apromoter is the promoter for the acid chitinase gene (Samac et al.,Plant Mol. Biol. 25:587-596 (1994)). Expression in root tissue couldalso be accomplished by utilizing the root specific subdomains of theCaMV35S promoter that have been identified (Lam et al., Proc. Natl Acad.Sci. (U.S.A.) 86:7890-7894 (1989)). Other root cell specific promotersinclude those reported by Conkling et al. (Conkling et al., PlantPhysiol. 93:1203-1211 (1990)).

[0235] Additional promoters that may be utilized are described, forexample, in U.S. Pat. Nos. 5,378,619; 5,391,725; 5,428,147; 5,447,858;5,608,144; 5,608,144; 5,614,399; 5,633,441; 5,633,435; and 4,633,436. Inaddition, a tissue specific enhancer may be used (Fromm et al., ThePlant Cell 1:977-984 (1989)).

[0236] In a preferred embodiment of the invention, a nucleic acidmolecule having a sequence encoding either a GMT or an MT1 enzyme islinked to a P7 or Arcelin 5 promoter. In a particularly preferredembodiment of the present invention, the promoter comprises a nucleicacid molecule having a sequence selected from the group consisting ofSEQ ID NOs 81 and 82. In a particularly preferred embodiment, theinvention relates to a soybean line 3244 plant, comprising an exogenousnucleic acid molecule comprising a nucleic acid sequence selected of SEQID NO: 2, operably linked to a nucleic acid molecule comprising anucleotide sequence selected from the group consisting of SEQ ID NO: 81and 82.

[0237] Constructs or vectors may also include, with the coding region ofinterest, a nucleic acid sequence that acts, in whole or in part, toterminate transcription of that region. A number of such sequences havebeen isolated, including the Tr7 3′ sequence and the NOS 3′ sequence(Ingelbrecht et al., The Plant Cell 1:671-680 (1989); Bevan et al.,Nucleic Acids Res. 11:369-385 (1983)). Regulatory transcript terminationregions can be provided in plant expression constructs of this inventionas well. Transcript termination regions can be provided by the DNAsequence encoding the gene of interest or a convenient transcriptiontermination region derived from a different gene source, for example,the transcript termination region that is naturally associated with thetranscript initiation region. The skilled artisan will recognize thatany convenient transcript termination region that is capable ofterminating transcription in a plant cell can be employed in theconstructs of the present invention.

[0238] A vector or construct may also include regulatory elements.Examples of such include the Adh intron 1 (Callis et al., Genes andDevelop. 1: 1183-1200 (1987)), the sucrose synthase intron (Vasil etal., Plant Physiol. 91:1575-1579 (1989)) and the TMV omega element(Gallie et al., The Plant Cell 1:301-311 (1989)). These and otherregulatory elements may be included when appropriate.

[0239] A vector or construct may also include a selectable marker.Selectable markers may also be used to select for plants or plant cellsthat contain the exogenous genetic material. Examples of such include,but are not limited to: a neo gene (Potrykus et al., Mol. Gen. Genet.199:183-188 (1985)), which codes for kanamycin resistance and can beselected for using kanamycin, RptII, G418, hpt etc.; a bar gene whichcodes for bialaphos resistance; a mutant EPSP synthase gene (Hinchee etal., Bio/Technology 6:915-922 (1988); Reynaerts et al., Selectable andScreenable Markers. In Gelvin and Schilperoort. Plant Molecular BiologyManual, Kluwer, Dordrecht (1988); Reynaerts et al., Selectable andscreenable markers. In Gelvin and Schilperoort. Plant Molecular BiologyManual, Kluwer, Dordrecht (1988)), aadA (Jones et al., Mol. Gen. Genet.(1987)),) which encodes glyphosate resistance; a nitrilase gene whichconfers resistance to bromoxynil (Stalker et al., J. Biol. Chem.263:6310-6314 (1988)); a mutant acetolactate synthase gene (ALS) whichconfers imidazolinone or sulphonylurea resistance (European PatentApplication 154,204 (Sep. 11, 1985)), ALS (D'Halluin et al.,Bio/Technology 10: 309-314 (1992)), and a methotrexate resistant DHFRgene (Thillet et al., J. Biol. Chem. 263:12500-12508 (1988)).

[0240] A vector or construct may also include a transit peptide.Incorporation of a suitable chloroplast transit peptide may also beemployed (European Patent Application Publication Number 0218571).Translational enhancers may also be incorporated as part of the vectorDNA. DNA constructs could contain one or more 5′ non-translated leadersequences, which may serve to enhance expression of the gene productsfrom the resulting mRNA transcripts. Such sequences may be derived fromthe promoter selected to express the gene or can be specificallymodified to increase translation of the mRNA. Such regions may also beobtained from viral RNAs, from suitable eukaryotic genes, or from asynthetic gene sequence. For a review of optimizing expression oftransgenes, see Koziel et al., Plant Mol. Biol. 32:393-405 (1996). Apreferred transit peptide is CTP1.

[0241] A vector or construct may also include a screenable marker.Screenable markers may be used to monitor expression. Exemplaryscreenable markers include: a β-glucuronidase or uidA gene (GUS) whichencodes an enzyme for which various chromogenic substrates are known(Jefferson, Plant Mol. Biol, Rep. 5:387-405 (1987); Jefferson et al.,EMBO J. 6:3901-3907 (1987)); an R-locus gene, which encodes a productthat regulates the production of anthocyanin pigments (red color) inplant tissues (Dellaporta et al., Stadler Symposium 11:263-282 (1988));a β-lactamase gene (Sutcliffe et al., Proc. Natl. Acad. Sci. (U.S.A.)75:3737-3741 (1978)), a gene which encodes an enzyme for which variouschromogenic substrates are known (e.g., PADAC, a chromogeniccephalosporin); a luciferase gene (Ow et al., Science 234:856-859(1986)); a xylE gene (Zukowsky et al., Proc. Natl. Acad. Sci. (U.S.A)80:1101-1105 (1983)) which encodes a catechol dioxygenase that canconvert chromogenic catechols; an α-amylase gene (Ikatu et al.,Bio/Technol. 8:241-242 (1990)); a tyrosinase gene (Katz et al., J. Gen.Microbiol. 129:2703-2714 (1983)) which encodes an enzyme capable ofoxidizing tyrosine to DOPA and dopaquinone which in turn condenses tomelanin; an α-galactosidase, which will turn a chromogenic α-galactosesubstrate.

[0242] Included within the terms “selectable or screenable marker genes”are also genes that encode a secretable marker whose secretion can bedetected as a means of identifying or selecting for transformed cells.Examples include markers that encode a secretable antigen that can beidentified by antibody interaction, or even secretable enzymes that canbe detected catalytically. Secretable proteins fall into a number ofclasses, including small, diffusible proteins that are detectable,(e.g., by ELISA), small active enzymes that are detectable inextracellular solution (e.g., α-amylase, β-lactamase, phosphinothricintransferase), or proteins that are inserted or trapped in the cell wall(such as proteins that include a leader sequence such as that found inthe expression unit of extension or tobacco PR-S). Other possibleselectable and/or screenable marker genes will be apparent to those ofskill in the art.

[0243] There are many methods for introducing transforming nucleic acidmolecules into plant cells. Suitable methods are believed to includevirtually any method by which nucleic acid molecules may be introducedinto a cell, such as by Agrobacterium infection or direct delivery ofnucleic acid molecules such as, for example, by PEG-mediatedtransformation, by electroporation or by acceleration of DNA coatedparticles, and the like. (Potrykus, Ann. Rev. Plant Physiol. Plant Mol.Biol. 42:205-225 (1991); Vasil, Plant Mol. Biol. 25:925-937 (1994)). Forexample, electroporation has been used to transform corn protoplasts(Fromm et al., Nature 312:791-793 (1986)).

[0244] Other vector systems suitable for introducing transforming DNAinto a host plant cell include but are not limited to binary artificialchromosome (BIBAC) vectors (Hamilton et al., Gene 200:107-116 (1997));and transfection with RNA viral vectors (Della-Cioppa et al., Ann. N.YAcad. Sci. (1996), 792 (Engineering Plants for Commercial Products andApplications), 57-61). Additional vector systems also include plantselectable YAC vectors such as those described in Mullen et al.,Molecular Breeding 4:449-457 (1988).

[0245] Technology for introduction of DNA into cells is well known tothose of skill in the art. Four general methods for delivering a geneinto cells have been described: (1) chemical methods (Graham and van derEb, Virology 54:536-539 (1973)); (2) physical methods such asmicroinjection (Capecchi, Cell 22:479-488 (1980)), electroporation (Wongand Neumann, Biochem. Biophys. Res. Commun. 107:584-587 (1982); Fromm etal., Proc. Natl. Acad. Sci. (U.S.A.) 82:5824-5828 (1985); U.S. Pat. No.5,384,253); the gene gun (Johnston and Tang, Methods Cell Biol.43:353-365 (1994)); and vacuum infiltration (Bechtold et al., C.R. Acad.Sci. Paris, Life Sci. 316:1194-1199. (1993)); (3) viral vectors (Clapp,Clin. Perinatol. 20:155-168 (1993); Lu et al., J. Exp. Med.178:2089-2096 (1993); Eglitis and Anderson, Biotechniques 6:608-614(1988)); and (4) receptor-mediated mechanisms (Curiel et al., Hum. Gen.Ther. 3:147-154 (1992), Wagner et al., Proc. Natl Acad. Sci. (USA)89:6099-6103 (1992)).

[0246] Acceleration methods that may be used include, for example,microprojectile bombardment and the like. One example of a method fordelivering transforming nucleic acid molecules into plant cells ismicroprojectile bombardment. This method has been reviewed by Yang andChristou (eds.), Particle Bombardment Technology for Gene Transfer,Oxford Press, Oxford, England (1994)). Non-biological particles(microprojectiles) may be coated with nucleic acids and delivered intocells by a propelling force. Exemplary particles include those comprisedof tungsten, gold, platinum and the like.

[0247] A particular advantage of microprojectile bombardment, inaddition to it being an effective means of reproducibly transformingmonocots, is that neither the isolation of protoplasts (Cristou et al.,Plant Physiol. 87:671-674 (1988)) nor the susceptibility toAgrobacterium infection is required. An illustrative embodiment of amethod for delivering DNA into corn cells by acceleration is abiolistics α-particle delivery system, which can be used to propelparticles coated with DNA through a screen, such as a stainless steel orNytex screen, onto a filter surface covered with corn cells cultured insuspension. Gordon-Kamm et al., describes the basic procedure forcoating tungsten particles with DNA (Gordon-Kamm et al., Plant Cell2:603-618 (1990)). The screen disperses the tungsten nucleic acidparticles so that they are not delivered to the recipient cells in largeaggregates. A particle delivery system suitable for use with theinvention is the helium acceleration PDS-1000/He gun, which is availablefrom Bio-Rad Laboratories (Bio-Rad, Hercules, Calif.)(Sanford et al.,Technique 3:3-16 (1991)).

[0248] For the bombardment, cells in suspension may be concentrated onfilters. Filters containing the cells to be bombarded are positioned atan appropriate distance below the microprojectile stopping plate. Ifdesired, one or more screens are also positioned between the gun and thecells to be bombarded.

[0249] Alternatively, immature embryos or other target cells may bearranged on solid culture medium. The cells to be bombarded arepositioned at an appropriate distance below the microprojectile stoppingplate. If desired, one or more screens are also positioned between theacceleration device and the cells to be bombarded. Through the use oftechniques set forth herein one may obtain 1000 or more loci of cellstransiently expressing a marker gene. The number of cells in a focusthat express the exogenous gene product 48 hours post-bombardment oftenranges from one to ten, and average one to three.

[0250] In bombardment transformation, one may optimize thepre-bombardment culturing conditions and the bombardment parameters toyield the maximum numbers of stable transformants. Both the physical andbiological parameters for bombardment are important in this technology.Physical factors are those that involve manipulating theDNA/microprojectile precipitate or those that affect the flight andvelocity of either the macro- or microprojectiles. Biological factorsinclude all steps involved in manipulation of cells before andimmediately after bombardment, the osmotic adjustment of target cells tohelp alleviate the trauma associated with bombardment and also thenature of the transforming DNA, such as linearized DNA or intactsupercoiled plasmids. It is believed that pre-bombardment manipulationsare especially important for successful transformation of immatureembryos.

[0251] In another alternative embodiment, plastids can be stablytransformed. Methods disclosed for plastid transformation in higherplants include the particle gun delivery of DNA containing a selectablemarker and targeting of the DNA to the plastid genome through homologousrecombination (Svab et al., Proc. Natl. Acad. Sci. (U.S.A.) 87:8526-8530(1990); Svab and Maliga, Proc. Natl. Acad. Sci. (U.S.A.) 90:913-917(1993); Staub and Maliga, EMBO J. 12:601-606 (1993); U.S. Pat. Nos.5,451,513 and 5,545,818).

[0252] Accordingly, it is contemplated that one may wish to adjustvarious aspects of the bombardment parameters in small scale studies tofully optimize the conditions. One may particularly wish to adjustphysical parameters such as gap distance, flight distance, tissuedistance and helium pressure. One may also minimize the trauma reductionfactors by modifying conditions that influence the physiological stateof the recipient cells and which may therefore influence transformationand integration efficiencies. For example, the osmotic state, tissuehydration and the subculture stage or cell cycle of the recipient cellsmay be adjusted for optimum transformation. The execution of otherroutine adjustments will be known to those of skill in the art in lightof the present disclosure.

[0253] Agrobacterium-mediated transfer is a widely applicable system forintroducing genes into plant cells because the DNA can be introducedinto whole plant tissues, thereby bypassing the need for regeneration ofan intact plant from a protoplast. The use of Agrobacterium-mediatedplant integrating vectors to introduce DNA into plant cells is wellknown in the art. See, for example the methods described by Fraley etal., Bio/Technology 3:629-635 (1985) and Rogers et al., Methods Enzymol.153:253-277 (1987). Further, the integration of the Ti-DNA is arelatively precise process resulting in few rearrangements. The regionof DNA to be transferred is defined by the border sequences andintervening DNA is usually inserted into the plant genome as described(Spielmann et al., Mol. Gen. Genet. 205:34 (1986)).

[0254] Modern Agrobacterium transformation vectors are capable ofreplication in E. coli as well as Agrobacterium, allowing for convenientmanipulations as described (Klee et al., In: Plant DNA InfectiousAgents, Hohn and Schell (eds.), Springer-Verlag, New York, pp. 179-203(1985)). Moreover, technological advances in vectors forAgrobacterium-mediated gene transfer have improved the arrangement ofgenes and restriction sites in the vectors to facilitate construction ofvectors capable of expressing various polypeptide coding genes. Thevectors described have convenient multi-linker regions flanked by apromoter and a polyadenylation site for direct expression of insertedpolypeptide coding genes and are suitable for present purposes (Rogerset al., Methods Enzymol. 153:253-277 (1987)). In addition, Agrobacteriumcontaining both armed and disarmed Ti genes can be used for thetransformations. In those plant strains where Agrobacterium-mediatedtransformation is efficient, it is the method of choice because of thefacile and defined nature of the gene transfer.

[0255] A transgenic plant formed using Agrobacterium transformationmethods typically contains a single gene on one chromosome. Suchtransgenic plants can be referred to as being heterozygous for the addedgene. More preferred is a transgenic plant that is homozygous for theadded structural gene; i.e., a transgenic plant that contains two addedgenes, one gene at the same locus on each chromosome of a chromosomepair. A homozygous transgenic plant can be obtained by sexually mating(selfing) an independent segregant, transgenic plant that contains asingle added gene, germinating some of the seed produced and analyzingthe resulting plants produced for the gene of interest.

[0256] It is also to be understood that two different transgenic plantscan also be mated to produce offspring that contain two independentlysegregating, exogenous genes. Selfing of appropriate progeny can produceplants that are homozygous for both added, exogenous genes that encode apolypeptide of interest. Back-crossing to a parental plant andout-crossing with a non-transgenic plant are also contemplated, as isvegetative propagation.

[0257] Transformation of plant protoplasts can be achieved using methodsbased on calcium phosphate precipitation, polyethylene glycol treatment,electroporation and combinations of these treatments (See, for example,Potrykus et al., Mol. Gen. Genet. 205:193-200 (1986); Lorz et al., Mol.Gen. Genet. 199:178 (1985); Fromm et al., Nature 319:791 (1986);Uchimiya et al., Mol. Gen. Genet. 204:204 (1986); Marcotte et al.,Nature 335:454-457 (1988)).

[0258] Application of these systems to different plant strains dependsupon the ability to regenerate that particular plant strain fromprotoplasts. Illustrative methods for the regeneration of cereals fromprotoplasts are described (Fujimura et al., Plant Tissue Culture Letters2:74 (1985); Toriyama et al., Theor. Appl. Genet. 205:34 (1986); Yamadaet al., Plant Cell Rep. 4:85 (1986); Abdullah et al., Biotechnology4:1087 (1986)).

[0259] To transform plant strains that cannot be successfullyregenerated from protoplasts, other ways to introduce DNA into intactcells or tissues can be utilized. For example, regeneration of cerealsfrom immature embryos or explants can be effected as described (Vasil,Biotechnology 6:397 (1988)). In addition, “particle gun” orhigh-velocity microprojectile technology can be utilized (Vasil et al.,Bio/Technology 10:667 (1992)).

[0260] Using the latter technology, DNA is carried through the cell walland into the cytoplasm on the surface of small metal particles asdescribed (Klein et al., Nature 328:70 (1987); Klein et al., Proc. Natl.Acad. Sci. (U.S.A.) 85:8502-8505 (1988); McCabe et al., Bio/Technology6:923 (1988)). The metal particles penetrate through several layers ofcells and thus allow the transformation of cells within tissue explants.

[0261] Other methods of cell transformation can also be used and includebut are not limited to introduction of DNA into plants by direct DNAtransfer into pollen (Hess et al., Intern Rev. Cytol. 107:367 (1987);Luo et al., Plant Mol Biol. Reporter 6:165 (1988)), by direct injectionof DNA into reproductive organs of a plant (Pena et al., Nature 325:274(1987)), or by direct injection of DNA into the cells of immatureembryos followed by the rehydration of desiccated embryos (Neuhaus etal., Theor. Appl. Genet. 75:30(1987)).

[0262] The regeneration, development and cultivation of plants fromsingle plant protoplast transformants or from various transformedexplants is well known in the art (Weissbach and Weissbach, In: Methodsfor Plant Molecular Biology, Academic Press, San Diego, Calif., (1988)).This regeneration and growth process typically includes the steps ofselection of transformed cells, culturing those individualized cellsthrough the usual stages of embryonic development through the rootedplantlet stage. Transgenic embryos and seeds are similarly regenerated.The resulting transgenic rooted shoots are thereafter planted in anappropriate plant growth medium such as soil.

[0263] The development or regeneration of plants containing the foreign,exogenous gene that encodes a protein of interest is well known in theart. Preferably, the regenerated plants are self-pollinated to providehomozygous transgenic plants. Otherwise, pollen obtained from theregenerated plants is crossed to seed-grown plants of agronomicallyimportant lines. Conversely, pollen from plants of these important linesis used to pollinate regenerated plants. A transgenic plant of theinvention containing a desired polypeptide is cultivated using methodswell known to one skilled in the art.

[0264] There are a variety of methods for the regeneration of plantsfrom plant tissue. The particular method of regeneration will depend onthe starting plant tissue and the particular plant species to beregenerated.

[0265] Methods for transforming dicots, primarily by use ofAgrobacterium tumefaciens and obtaining transgenic plants have beenpublished for cotton (U.S. Pat. No. 5,004,863; U.S. Pat. No. 5,159,135;U.S. Pat. No. 5,518,908); soybean (U.S. Pat. No. 5,569,834; U.S. Pat.No. 5,416,011; McCabe et al., Biotechnology 6:923 (1988); Christou etal., Plant Physiol. 87:671-674 (1988)); Brassica (U.S. Pat. No.5,463,174); peanut (Cheng et al., Plant Cell Rep. 15:653-657 (1996),McKently et al., Plant Cell Rep. 14:699-703 (1995)); papaya; pea (Grantet al., Plant Cell Rep. 15:254-258 (1995)); and Arabidopsis thaliana(Bechtold et al., C.R. Acad. Sci. Paris, Life Sci. 316:1194-1199(1993)). The latter method for transforming Arabidopsis thaliana iscommonly called “dipping” or vacuum infiltration or germplasmtransformation.

[0266] Transformation of monocotyledons using electroporation, particlebombardment and Agrobacterium have also been reported. Transformationand plant regeneration have been achieved in asparagus (Bytebier et al.,Proc. Natl Acad. Sci. (USA) 84:5354 (1987)); barley (Wan and Lemaux,Plant Physiol 104:37 (1994)); corn (Rhodes et al., Science 240:204(1988); Gordon-Kamm et al., Plant Cell 2:603-618 (1990); Fromm et al.,Bio/Technology 8:833 (1990); Koziel et al., Bio/Technology 11:194(1993); Armstrong et al., Crop Science 35:550-557 (1995)); oat (Somerset al., Bio/Technology 10:1589 (1992)); orchard grass (Horn et al.,Plant Cell Rep. 7:469 (1988)); rice (Toriyama et al., Theor Appl. Genet.205:34 (1986); Part et al., Plant Mol. Biol. 32:1135-1148 (1996);Abedinia et al., Aust. J. Plant Physiol. 24:133-141 (1997); Zhang andWu, Theor. Appl. Genet. 76:835 (1988); Zhang et al., Plant Cell Rep.7:379 (1988); Battraw and Hall, Plant Sci. 86:191-202 (1992); Christouet al., Bio/Technology 9:957 (1991)); rye (De la Pena et al., Nature325:274 (1987)); sugarcane (Bower and Birch, Plant J. 2:409 (1992));tall fescue (Wang et al., Bio/Technology 10:691 (1992)) and wheat (Vasilet al., Bio/Technology 10:667 (1992); U.S. Pat. No. 5,631,152).

[0267] Assays for gene expression based on the transient expression ofcloned nucleic acid constructs have been developed by introducing thenucleic acid molecules into plant cells by polyethylene glycoltreatment, electroporation, or particle bombardment (Marcotte et al.,Nature 335:454-457 (1988); Marcotte et al., Plant Cell 1:523-532 (1989);McCarty et al., Cell 66:895-905 (1991); Hattori et al., Genes Dev.6:609-618 (1992); Goff et al., EMBO J. 9:2517-2522 (1990)). Transientexpression systems may be used to functionally dissect gene constructs(see generally, Mailga et al., Methods in Plant Molecular Biology, ColdSpring Harbor Press (1995)).

[0268] Any of the nucleic acid molecules of the invention may beintroduced into a plant cell in a permanent or transient manner incombination with other genetic elements such as vectors, promoters,enhancers, etc. Further, any of the nucleic acid molecules of theinvention may be introduced into a plant cell in a manner that allowsfor expression or overexpression of the protein or fragment thereofencoded by the nucleic acid molecule.

[0269] Cosuppression is the reduction in expression levels, usually atthe level of RNA, of a particular endogenous gene or gene family by theexpression of a homologous sense construct that is capable oftranscribing mRNA of the same strandedness as the transcript of theendogenous gene (Napoli et al., Plant Cell 2:279-289 (1990); van derKrol et al., Plant Cell 2:291-299 (1990)). Cosuppression may result fromstable transformation with a single copy nucleic acid molecule that ishomologous to a nucleic acid sequence found with the cell (Prolls andMeyer, Plant J. 2:465-475 (1992)) or with multiple copies of a nucleicacid molecule that is homologous to a nucleic acid sequence found withthe cell (Mittlesten et al., Mol Gen. Genet. 244.325-330 (1994)). Genes,even though different, linked to homologous promoters may result in thecosuppression of the linked genes (Vaucheret, C. R. Acad. Sci.III316:1471-1483 (1993); Flavell, Proc. Natl. Acad. Sci. (U.S.A.)91:3490-3496 (1994)); van Blokland et al., Plant J. 6:861-877 (1994);Jorgensen, Trends Biotechnol. 8:340-344 (1990); Meins and Kunz, In: GeneInactivation and Homologous Recombination in Plants, Paszkowski (ed.),pp. 335-348, Kluwer Academic, Netherlands (1994)).

[0270] It is understood that one or more of the nucleic acids of theinvention may be introduced into a plant cell and transcribed using anappropriate promoter with such transcription resulting in thecosuppression of an endogenous protein.

[0271] Antisense approaches are a way of preventing or reducing genefunction by targeting the genetic material (Mol et al., FEBS Lett.268:427-430 (1990)). The objective of the antisense approach is to use asequence complementary to the target gene to block its expression andcreate a mutant cell line or organism in which the level of a singlechosen protein is selectively reduced or abolished. Antisense techniqueshave several advantages over other ‘reverse genetic’ approaches. Thesite of inactivation and its developmental effect can be manipulated bythe choice of promoter for antisense genes or by the timing of externalapplication or microinjection. Antisense can manipulate its specificityby selecting either unique regions of the target gene or regions whereit shares homology to other related genes (Hiatt et al., In: GeneticEngineering, Setlow (ed.), Vol. 11, New York: Plenum 49-63 (1989)).

[0272] Antisense RNA techniques involve introduction of RNA that iscomplementary to the target mRNA into cells, which results in specificRNA:RNA duplexes being formed by base pairing between the antisensesubstrate and the target mRNA (Green et al, Annu. Rev. Biochem.55:569-597 (1986)). Under one embodiment, the process involves theintroduction and expression of an antisense gene sequence. Such asequence is one in which part or all of the normal gene sequences areplaced under a promoter in inverted orientation so that the ‘wrong’ orcomplementary strand is transcribed into a noncoding antisense RNA thathybridizes with the target mRNA and interferes with its expression(Takayama and Inouye, Crit. Rev. Biochem. Mol. Biol. 25:155-184 (1990)).An antisense vector is constructed by standard procedures and introducedinto cells by transformation, transfection, electroporation,microinjection, infection, etc. The type of transformation and choice ofvector will determine whether expression is transient or stable. Thepromoter used for the antisense gene may influence the level, timing,tissue, specificity, or inducibility of the antisense inhibition.

[0273] It is understood that the activity of a protein in a plant cellmay be reduced or depressed by growing a transformed plant cellcontaining a nucleic acid molecule whose non-transcribed strand encodesa protein or fragment thereof. Preferred proteins whose activity can bereduced or depressed, by any method, are MT1 and homogenistic aciddehydrogenase. In such an embodiment of the invention, it is preferredthat the concentration of γ-tocopherol or γ-tocotrienol is increased.

[0274] Posttranscriptional gene silencing (PTGS) can result in virusimmunity or gene silencing in plants. PTGS is induced by dsRNA and ismediated by an RNA-dependent RNA polymerase, present in the cytoplasm,which requires a dsRNA template. The dsRNA is formed by hybridization ofcomplementary transgene mRNAs or complementary regions of the sametranscript. Duplex formation can be accomplished by using transcriptsfrom one sense gene and one antisense gene colocated in the plantgenome, a single transcript that has self-complementarity, or sense andantisense transcripts from genes brought together by crossing. ThedsRNA-dependent RNA polymerase makes a complementary strand from thetransgene mRNA and RNAse molecules attach to this complementary strand(cRNA). These cRNA-RNase molecules hybridize to the endogene mRNA andcleave the single-stranded RNA adjacent to the hybrid. The cleavedsingle-stranded RNAs are further degraded by other host RNases becauseone will lack a capped 5′ end and the other will lack a poly(A) tail(Waterhouse et al., PNAS 95: 13959-13964 (1998)).

[0275] It is understood that one or more of the nucleic acids of theinvention may be introduced into a plant cell and transcribed using anappropriate promoter with such transcription resulting in theposttranscriptional gene silencing of an endogenous transcript.

[0276] Antibodies have been expressed in plants (Hiatt et al., Nature342:76-78 (1989); Conrad and Fielder, Plant Mol. Biol. 26:1023-1030(1994)). Cytoplasmic expression of a scFv (single-chain Fv antibody) hasbeen reported to delay infection by artichoke mottled crinkle virus.Transgenic plants that express antibodies directed against endogenousproteins may exhibit a physiological effect (Philips et al., EMBO J.16:4489-4496 (1997); Marion-Poll, Trends in Plant Science 2:447-448(1997)). For example, expressed anti-abscisic antibodies have beenreported to result in a general perturbation of seed development(Philips et al., EMBO J. 16: 4489-4496 (1997)).

[0277] Antibodies that are catalytic may also be expressed in plants(abzymes). The principle behind abzymes is that since antibodies may beraised against many molecules, this recognition ability can be directedtoward generating antibodies that bind transition states to force achemical reaction forward (Persidas, Nature Biotechnology 15:1313-1315(1997); Baca et al., Ann. Rev. Biophys. Biomol. Struct. 26:461-493(1997)). The catalytic abilities of abzymes may be enhanced by sitedirected mutagenesis. Examples of abzymes are, for example, set forth inU.S. Pat. No. 5,658,753; U.S. Pat. No. 5,632,990; U.S. Pat. No.5,631,137; U.S. Pat. No. 5,602,015; U.S. Pat. No. 5,559,538; U.S. Pat.No. 5,576,174; U.S. Pat. No. 5,500,358; U.S. Pat. No. 5,318,897; U.S.Pat. No. 5,298,409; U.S. Pat. No. 5,258,289 and U.S. Pat. No. 5,194,585.

[0278] It is understood that any of the antibodies of the invention maybe expressed in plants and that such expression can result in aphysiological effect. It is also understood that any of the expressedantibodies may be catalytic.

[0279] The present invention also provides for parts of the plants,particularly reproductive or storage parts, of the present invention.Plant parts, without limitation, include seed, endosperm, ovule andpollen. In a particularly preferred embodiment of the present invention,the plant part is a seed. In one embodiment the seed is a constituent ofanimal feed.

[0280] In another embodiment, the plant part is a fruit, more preferablya fruit with enhanced shelf life. In another preferred embodiment, thefruit has increased levels of a tocopherol. In another preferredembodiment, the fruit has increased levels of a tocotrienol.

[0281] The present invention also provides a container of over about10,000, more preferably about 20,000, and even more preferably about40,000 seeds where over about 10%, more preferably about 25%, morepreferably about 50% and even more preferably about 75% or 90% of theseeds are seeds derived from a plant of the present invention.

[0282] The present invention also provides a container of over about 10kg, more preferably about 25 kg, and even more preferably about 50 kgseeds where over about 10%, more preferably about 25%, more preferablyabout 50% and even more preferably about 75% or 90% of the seeds areseeds derived from a plant of the present invention.

[0283] Any of the plants or parts thereof of the present invention maybe processed to produce a feed, meal, protein or oil preparation. Aparticularly preferred plant part for this purpose is a seed. In apreferred embodiment the feed, meal, protein or oil preparation isdesigned for ruminant animals. Methods to produce feed, meal, proteinand oil preparations are known in the art. See, for example, U.S. Pat.Nos. 4,957,748, 5,100,679, 5,219,596, 5,936,069, 6,005,076, 6,146,669,and 6,156,227. In a preferred embodiment, the protein preparation is ahigh protein preparation. Such a high protein preparation preferably hasa protein content of greater than 5% w/v, more preferably 10% w/v, andeven more preferably 15% w/v. In a preferred oil preparation, the oilpreparation is a high oil preparation with an oil content derived from aplant or part thereof of the present invention of greater than 5% w/v,more preferably 10% w/v, and even more preferably 15% w/v. In apreferred embodiment the oil preparation is a liquid and of a volumegreater than 1, 5, 10 or 50 liters. The present invention provides foroil produced from plants of the present invention or generated by amethod of the present invention. Such an oil may exhibit enhancedoxidative stability. Also, such oil may be a minor or major component ofany resultant product. Moreover, such oil may be blended with otheroils. In a preferred embodiment, the oil produced from plants of thepresent invention or generated by a method of the present inventionconstitutes greater than 0.5%, 1%, 5%, 10%, 25%, 50%, 75% or 90% byvolume or weight of the oil component of any product. In anotherembodiment, the oil preparation may be blended and can constitutegreater than 10%, 25%, 35%, 50% or 75% of the blend by volume. Oilproduced from a plant of the present invention can be admixed with oneor more organic solvents or petroleum distillates.

[0284] Plants of the present invention can be part of or generated froma breeding program. The choice of breeding method depends on the mode ofplant reproduction, the heritability of the trait(s) being improved, andthe type of cultivar used commercially (e.g., F₁ hybrid cultivar,pureline cultivar, etc). Selected, non-limiting approaches, for breedingthe plants of the present invention are set forth below. A breedingprogram can be enhanced using marker assisted selection of the progenyof any cross. It is further understood that any commercial andnon-commercial cultivars can be utilized in a breeding program. Factorssuch as, for example, emergence vigor, vegetative vigor, stresstolerance, disease resistance, branching, flowering, seed set, seedsize, seed density, standability, and threshability etc. will generallydictate the choice.

[0285] For highly heritable traits, a choice of superior individualplants evaluated at a single location will be effective, whereas fortraits with low heritability, selection should be based on mean valuesobtained from replicated evaluations of families of related plants.Popular selection methods commonly include pedigree selection, modifiedpedigree selection, mass selection, and recurrent selection. In apreferred embodiment a backcross or recurrent breeding program isundertaken.

[0286] The complexity of inheritance influences choice of the breedingmethod. Backcross breeding can be used to transfer one or a fewfavorable genes for a highly heritable trait into a desirable cultivar.This approach has been used extensively for breeding disease-resistantcultivars. Various recurrent selection techniques are used to improvequantitatively inherited traits controlled by numerous genes. The use ofrecurrent selection in self-pollinating crops depends on the ease ofpollination, the frequency of successful hybrids from each pollination,and the number of hybrid offspring from each successful cross.

[0287] Breeding lines can be tested and compared to appropriatestandards in environments representative of the commercial targetarea(s) for two or more generations. The best lines are candidates fornew commercial cultivars; those still deficient in traits may be used asparents to produce new populations for further selection.

[0288] One method of identifying a superior plant is to observe itsperformance relative to other experimental plants and to a widely grownstandard cultivar. If a single observation is inconclusive, replicatedobservations can provide a better estimate of its genetic worth. Abreeder can select and cross two or more parental lines, followed byrepeated selfing and selection, producing many new genetic combinations.

[0289] The development of new cultivars requires the development andselection of varieties, the crossing of these varieties and theselection of superior hybrid crosses. The hybrid seed can be produced bymanual crosses between selected male-fertile parents or by using malesterility systems. Hybrids are selected for certain single gene traitssuch as pod color, flower color, seed yield, pubescence color, orherbicide resistance, which indicate that the seed is truly a hybrid.Additional data on parental lines, as well as the phenotype of thehybrid, influence the breeder's decision whether to continue with thespecific hybrid cross.

[0290] Pedigree breeding and recurrent selection breeding methods can beused to develop cultivars from breeding populations. Breeding programscombine desirable traits from two or more cultivars or variousbroad-based sources into breeding pools from which cultivars aredeveloped by selfing and selection of desired phenotypes. New cultivarscan be evaluated to determine which have commercial potential.

[0291] Pedigree breeding is used commonly for the improvement ofself-pollinating crops. Two parents who possess favorable, complementarytraits are crossed to produce an F₁. A F₂ population is produced byselfing one or several F₁'s. Selection of the best individuals from thebest families is carried out. Replicated testing of families can beginin the F₄ generation to improve the effectiveness of selection fortraits with low heritability. At an advanced stage of inbreeding (i.e.,F₆ and F₇), the best lines or mixtures of phenotypically similar linesare tested for potential release as new cultivars.

[0292] Backcross breeding has been used to transfer genes for a simplyinherited, highly heritable trait into a desirable homozygous cultivaror inbred line, which is the recurrent parent. The source of the traitto be transferred is called the donor parent. The resulting plant isexpected to have the attributes of the recurrent parent (e.g., cultivar)and the desirable trait transferred from the donor parent. After theinitial cross, individuals possessing the phenotype of the donor parentare selected and repeatedly crossed (back-crossed) to the recurrentparent. The resulting parent is expected to have the attributes of therecurrent parent (e.g., cultivar) and the desirable trait transferredfrom the donor parent.

[0293] The single-seed descent procedure in the strict sense refers toplanting a segregating population, harvesting a sample of one seed perplant, and using the one-seed sample to plant the next generation. Whenthe population has been advanced from the F₂ to the desired level ofinbreeding, the plants from which lines are derived will each trace todifferent F₂ individuals. The number of plants in a population declineseach generation due to failure of some seeds to germinate or some plantsto produce at least one seed. As a result, not all of the F₂ plantsoriginally sampled in the population will be represented by a progenywhen generation advance is completed.

[0294] In a multiple-seed procedure, breeders commonly harvest one ormore pods from each plant in a population and thresh them together toform a bulk. Part of the bulk is used to plant the next generation andpart is put in reserve. The procedure has been referred to as modifiedsingle-seed descent or the pod-bulk technique.

[0295] The multiple-seed procedure has been used to save labor atharvest. It is considerably faster to thresh pods with a machine than toremove one seed from each by hand for the single-seed procedure. Themultiple-seed procedure also makes it possible to plant the same numberof seed of a population each generation of inbreeding.

[0296] Descriptions of other breeding methods that are commonly used fordifferent traits and crops can be found in one of several referencebooks (e.g. Fehr, Principles of Cultivar Development Vol. 1, pp. 2-3(1987))).

[0297] A transgenic plant of the present invention may also bereproduced using apomixis. Apomixis is a genetically controlled methodof reproduction in plants where the embryo is formed without union of anegg and a sperm. There are three basic types of apomicticreproduction: 1) apospory where the embryo develops from a chromosomallyunreduced egg in an embryo sac derived from the nucleus, 2) diplosporywhere the embryo develops from an unreduced egg in an embryo sac derivedfrom the megaspore mother cell, and 3) adventitious embryony where theembryo develops directly from a somatic cell. In most forms of apomixis,pseudogamy or fertilization of the polar nuclei to produce endosperm isnecessary for seed viability. In apospory, a nurse cultivar can be usedas a pollen source for endosperm formation in seeds. The nurse cultivardoes not affect the genetics of the aposporous apomictic cultivar sincethe unreduced egg of the cultivar develops parthenogenetically, butmakes possible endosperm production. Apomixis is economically important,especially in transgenic plants, because it causes any genotype, nomatter how heterozygous, to breed true. Thus, with apomicticreproduction, heterozygous transgenic plants can maintain their geneticfidelity throughout repeated life cycles. Methods for the production ofapomictic plants are known in the art. See, U.S. Pat. No. 5,811,636.

[0298] Other Organisms

[0299] A nucleic acid of the present invention may be introduced intoany cell or organism such as a mammalian cell, mammal, fish cell, fish,bird cell, bird, algae cell, algae, fungal cell, fungi, or bacterialcell. A protein of the present invention may be produced in anappropriate cell or organism. Preferred host and transformants include:fungal cells such as Aspergillus, yeasts, mammals, particularly bovineand porcine, insects, bacteria, and algae. Particularly preferredbacteria are agrobacterium tumefaciens and E. coli.

[0300] Methods to transform such cells or organisms are known in the art(EP 0 238 023; Yelton et al., Proc. Natl. Acad. Sci. (U.S.A.),81:1470-1474 (1984); Malardier et al., Gene, 78:147-156 (1989); Beckerand Guarente, In: Abelson and Simon (eds.), Guide to Yeast Genetics andMolecular Biology, Method Enzymol., Vol. 194, pp. 182-187, AcademicPress, Inc., New York; Ito et al., J. Bacteriology, 153:163 (1983)Hinnen et al., Proc. Natl. Acad. Sci. (U.S.A.), 75:1920 (1978); Bennettand LaSure (eds.), More Gene Manipulation in fungi, Academic Press, CA(1991)). Methods to produce proteins of the present invention are alsoknown (Kudla et al., EMBO, 9:1355-1364 (1990); Jarai and Buxton, CurrentGenetics, 26:2238-2244 (1994); Verdier, Yeast, 6:271-297 (1990;MacKenzie et al., Journal of Gen. Microbiol., 139:2295-2307 (1993);Hartl et al., TIBS, 19:20-25 (1994); Bergenron et al., TIBS, 19:124-128(1994); Demolder et al., J. Biotechnology, 32:179-189 (1994); Craig,Science, 260:1902-1903 (1993); Gething and Sambrook, Nature, 355:33-45(1992); Puig and Gilbert, J. Biol. Chem., 269:7764-7771 (1994); Wang andTsou, FASEB Journal, 7:1515-1517 (1993); Robinson et al.,Bio/Technology, 1:381-384 (1994); Enderlin and Ogrydziak, Yeast,10:67-79 (1994); Fuller et al., Proc. Natl. Acad. Sci. (U.S.A.),86:1434-1438 (1989); Julius et al., Cell, 37:1075-1089 (1984); Julius etal., Cell 32:839-852 (1983).

[0301] In a preferred embodiment, overexpression of a protein orfragment thereof of the present invention in a cell or organism providesin that cell or organism, relative to an untransformed cell or organismwith a similar genetic background, an increased level of tocopherols.

[0302] In a preferred embodiment, overexpression of a protein orfragment thereof of the present invention in a cell or organism providesin that cell or organism, relative to an untransformed cell or organismwith a similar genetic background, an increased level of α-tocopherols.

[0303] In a preferred embodiment, overexpression of a protein orfragment thereof of the present invention in a cell or organism providesin that cell or organism, relative to an untransformed cell or organismwith a similar genetic background, an increased level of γ-tocopherols.

[0304] In another preferred embodiment, overexpression of a protein orfragment thereof of the present invention in a cell or organism providesin that cell or organism, relative to an untransformed cell or organismwith a similar genetic background, an increased level of α-tocotrienols.

[0305] In another preferred embodiment, overexpression of a protein orfragment thereof of the present invention in a cell or organism providesin that cell or organism, relative to an untransformed cell or organismwith a similar genetic background, an increased level of γ-tocotrienols.

[0306] Antibodies

[0307] One aspect of the invention concerns antibodies, single-chainantigen binding molecules, or other proteins that specifically bind toone or more of the protein or peptide molecules of the invention andtheir homologs, fusions or fragments. In a particularly preferredembodiment, the antibody specifically binds to a protein having theamino acid sequence set forth in SEQ ID NOs: 19-31, 33-38, 39-41, and46-49 or a fragment thereof. In another embodiment, the antibodyspecifically binds to a fusion protein comprising an amino acid sequenceselected from the amino acid sequence set forth in SEQ ID NOs: 19-33 and33-38 or a fragment thereof. In another embodiment the antibodyspecifically binds to a fusion protein comprising an amino acid sequenceselected from the amino acid sequence set forth in SEQ ID NOs: 46-49 ora fragment thereof. Antibodies of the invention may be used toquantitatively or qualitatively detect the protein or peptide moleculesof the invention, or to detect post translational modifications of theproteins. As used herein, an antibody or peptide is said to“specifically bind” to a protein or peptide molecule of the invention ifsuch binding is not competitively inhibited by the presence ofnon-related molecules.

[0308] Nucleic acid molecules that encode all or part of the protein ofthe invention can be expressed, via recombinant means, to yield proteinor peptides that can in turn be used to elicit antibodies that arecapable of binding the expressed protein or peptide. Such antibodies maybe used in immunoassays for that protein. Such protein-encodingmolecules, or their fragments may be a “fusion” molecule (i.e., a partof a larger nucleic acid molecule) such that, upon expression, a fusionprotein is produced. It is understood that any of the nucleic acidmolecules of the invention may be expressed, via recombinant means, toyield proteins or peptides encoded by these nucleic acid molecules.

[0309] The antibodies that specifically bind proteins and proteinfragments of the invention may be polyclonal or monoclonal and maycomprise intact immunoglobulins, or antigen binding portions ofimmunoglobulins fragments (such as (F(ab′), F(ab′)₂), or single-chainimmunoglobulins producible, for example, via recombinant means. It isunderstood that practitioners are familiar with the standard resourcematerials that describe specific conditions and procedures for theconstruction, manipulation and isolation of antibodies (see, forexample, Harlow and Lane, In: Antibodies: A Laboratory Manual, ColdSpring Harbor Press, Cold Spring Harbor, N.Y. (1988)).

[0310] As discussed below, such antibody molecules or their fragmentsmay be used for diagnostic purposes. Where the antibodies are intendedfor diagnostic purposes, it may be desirable to derivatize them, forexample with a ligand group (such as biotin) or a detectable markergroup (such as a fluorescent group, a radioisotope or an enzyme).

[0311] The ability to produce antibodies that bind the protein orpeptide molecules of the invention permits the identification of mimeticcompounds derived from those molecules. These mimetic compounds maycontain a fragment of the protein or peptide or merely a structurallysimilar region and nonetheless exhibits an ability to specifically bindto antibodies directed against that compound.

[0312] Exemplary Uses

[0313] Nucleic acid molecules and fragments thereof of the invention maybe employed to obtain other nucleic acid molecules from the same species(nucleic acid molecules from corn may be utilized to obtain othernucleic acid molecules from corn). Such nucleic acid molecules includethe nucleic acid molecules that encode the complete coding sequence of aprotein and promoters and flanking sequences of such molecules. Inaddition, such nucleic acid molecules include nucleic acid moleculesthat encode for other isozymes or gene family members. Such moleculescan be readily obtained by using the above-described nucleic acidmolecules or fragments thereof to screen cDNA or genomic libraries.Methods for forming such libraries are well known in the art.

[0314] Nucleic acid molecules and fragments thereof of the invention mayalso be employed to obtain nucleic acid homologs. Such homologs includethe nucleic acid molecules of plants and other organisms, includingbacteria and fungi, including the nucleic acid molecules that encode, inwhole or in part, protein homologues of other plant species or otherorganisms, sequences of genetic elements, such as promoters andtranscriptional regulatory elements. Such molecules can be readilyobtained by using the above-described nucleic acid molecules orfragments thereof to screen cDNA or genomic libraries obtained from suchplant species. Methods for forming such libraries are well known in theart. Such homolog molecules may differ in their nucleotide sequencesfrom those found in one or more of SEQ ID NOs: 2-17, 50, and 85 andcomplements thereof because complete complementarity is not needed forstable hybridization. The nucleic acid molecules of the inventiontherefore also include molecules that, although capable of specificallyhybridizing with the nucleic acid molecules may lack “completecomplementarity.”

[0315] Any of a variety of methods may be used to obtain one or more ofthe above-described nucleic acid molecules (Zamechik et al., Proc. Natl.Acad. Sci. (U.S.A.) 83:4143-4146 (1986); Goodchild et al., Proc. Natl.Acad. Sci. (U.S.A.) 85:5507-5511 (1988); Wickstrom et al., Proc. Natl.Acad. Sci. (U.S.A.) 85:1028-1032 (1988); Holt et al., Molec. Cell. Biol.8:963-973 (1988); Gerwirtz et al., Science 242:1303-1306 (1988); Anfossiet al., Proc. Natl. Acad. Sci. (U.S.A.) 86:3379-3383 (1989); Becker etal., EMBO J. 8:3685-3691 (1989)). Automated nucleic acid synthesizersmay be employed for this purpose. In lieu of such synthesis, thedisclosed nucleic acid molecules may be used to define a pair of primersthat can be used with the polymerase chain reaction (Mullis et al., ColdSpring Harbor Symp. Quant. Biol. 51:263-273 (1986); Erlich et al.,European Patent 50,424; European Patent 84,796; European Patent 258,017;European Patent 237,362; Mullis, European Patent 201,184; Mullis et al.,U.S. Pat. No. 4,683,202; Erlich, U.S. Pat. No. 4,582,788; and Saiki etal., U.S. Pat. No. 4,683,194) to amplify and obtain any desired nucleicacid molecule or fragment.

[0316] Promoter sequences and other genetic elements, including but notlimited to transcriptional regulatory flanking sequences, associatedwith one or more of the disclosed nucleic acid sequences can also beobtained using the disclosed nucleic acid sequence provided herein. Inone embodiment, such sequences are obtained by incubating nucleic acidmolecules of the present invention with members of genomic libraries andrecovering clones that hybridize to such nucleic acid molecules thereof.In a second embodiment, methods of “chromosome walking,” or inverse PCRmay be used to obtain such sequences (Frohman et al., Proc. Natl. Acad.Sci. (U.S.A.) 85:8998-9002 (1988); Ohara et al., Proc. Natl. Acad. Sci.(U.S.A.) 86:5673-5677 (1989); Pang et al., Biotechniques 22:1046-1048(1977); Huang et al., Methods Mol. Biol. 69:89-96 (1997); Huang et al.,Method Mol. Biol. 67:287-294 (1997); Benkel et al., Genet. Anal.13:123-127 (1996); Hartl et al., Methods Mol. Biol. 58:293-301 (1996)).The term “chromosome walking” means a process of extending a genetic mapby successive hybridization steps.

[0317] The nucleic acid molecules of the invention may be used toisolate promoters of cell enhanced, cell specific, tissue enhanced,tissue specific, developmentally or environmentally regulated expressionprofiles. Isolation and functional analysis of the 5′ flanking promotersequences of these genes from genomic libraries, for example, usinggenomic screening methods and PCR techniques would result in theisolation of useful promoters and transcriptional regulatory elements.These methods are known to those of skill in the art and have beendescribed (See, for example, Birren et al., Genome Analysis: AnalyzingDNA, 1, (1997), Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y.). Promoters obtained utilizing the nucleic acid molecules of theinvention could also be modified to affect their controlcharacteristics. Examples of such modifications would include but arenot limited to enhancer sequences. Such genetic elements could be usedto enhance gene expression of new and existing traits for cropimprovement.

[0318] Another subset of the nucleic acid molecules of the inventionincludes nucleic acid molecules that are markers. The markers can beused in a number of conventional ways in the field of moleculargenetics. Such markers include nucleic acid molecules SEQ ID NOs: 2-17,50, and 85, complements thereof, and fragments of either that can act asmarkers and other nucleic acid molecules of the present invention thatcan act as markers.

[0319] Genetic markers of the invention include “dominant” or“codominant” markers. “Codominant markers” reveal the presence of two ormore alleles (two per diploid individual) at a locus. “Dominant markers”reveal the presence of only a single allele per locus. The presence ofthe dominant marker phenotype (e.g., a band of DNA) is an indicationthat one allele is in either the homozygous or heterozygous condition.The absence of the dominant marker phenotype (e.g., absence of a DNAband) is merely evidence that “some other” undefined allele is present.In the case of populations where individuals are predominantlyhomozygous and loci are predominately dimorphic, dominant andco-dominant markers can be equally valuable. As populations become moreheterozygous and multi-allelic, codominant markers often become moreinformative of the genotype than dominant markers. Marker molecules canbe, for example, capable of detecting polymorphisms such as singlenucleotide polymorphisms (SNPs).

[0320] The genomes of animals and plants naturally undergo spontaneousmutation in the course of their continuing evolution (Gusella, Ann. Rev.Biochem. 55:831-854 (1986)). A “polymorphism” is a variation ordifference in the sequence of the gene or its flanking regions thatarises in some of the members of a species. The variant sequence and the“original” sequence co-exist in the species' population. In someinstances, such co-existence is in stable or quasi-stable equilibrium.

[0321] A polymorphism is thus said to be “allelic,” in that, due to theexistence of the polymorphism, some members of a population may have theoriginal sequence (i.e., the original “allele”) whereas other membersmay have the variant sequence (i.e., the variant “allele”). In thesimplest case, only one variant sequence may exist and the polymorphismis thus said to be di-allelic. In other cases, the species' populationmay contain multiple alleles and the polymorphism is termed tri-allelic,etc. A single gene may have multiple different unrelated polymorphisms.For example, it may have a di-allelic polymorphism at one site and amulti-allelic polymorphism at another site.

[0322] The variation that defines the polymorphism may range from asingle nucleotide variation to the insertion or deletion of extendedregions within a gene. In some cases, the DNA sequence variations are inregions of the genome that are characterized by short tandem repeats(STRs) that include tandem di- or tri-nucleotide repeated motifs ofnucleotides. Polymorphisms characterized by such tandem repeats arereferred to as “variable number tandem repeat” (“VNTR”) polymorphisms.VNTRs have been used in identity analysis (Weber, U.S. Pat. No.5,075,217; Armour et al., FEBS Lett. 307:113-115 (1992); Jones et al.,Eur. J. Haematol. 39:144-147 (1987); Horn et al., PCT Patent ApplicationWO 91/14003; Jeffreys, European Patent Application 370,719; Jeffreys,U.S. Pat. No. 5,175,082; Jeffreys et al., Amer. J. Hum. Genet. 39:11-24(1986); Jeffreys et al., Nature 316:76-79 (1985); Gray et al., Proc. R.Acad. Soc. Lond. 243:241-253 (1991); Moore et al., Genomics 10:654-660(1991); Jeffreys et al., Anim. Genet. 18:1-15 (1987); Hillel et al.,Anim. Genet. 20:145-155 (1989); Hillel et al., Genet. 124:783-789(1990)).

[0323] The detection of polymorphic sites in a sample of DNA may befacilitated through the use of nucleic acid amplification methods. Suchmethods specifically increase the concentration of polynucleotides thatspan the polymorphic site, or include that site and sequences locatedeither distal or proximal to it. Such amplified molecules can be readilydetected by gel electrophoresis or other means.

[0324] In an alternative embodiment, such polymorphisms can be detectedthrough the use of a marker nucleic acid molecule that is physicallylinked to such polymorphism(s). For this purpose, marker nucleic acidmolecules comprising a nucleotide sequence of a polynucleotide locatedwithin 1 mb of the polymorphism(s) and more preferably within 100 kb ofthe polymorphism(s) and most preferably within 10 kb of thepolymorphism(s) can be employed.

[0325] The identification of a polymorphism can be determined in avariety of ways. By correlating the presence or absence of it in a plantwith the presence or absence of a phenotype, it is possible to predictthe phenotype of that plant. If a polymorphism creates or destroys arestriction endonuclease cleavage site, or if it results in the loss orinsertion of DNA (e.g., a VNTR polymorphism), it will alter the size orprofile of the DNA fragments that are generated by digestion with thatrestriction endonuclease. As such, organisms that possess a variantsequence can be distinguished from those having the original sequence byrestriction fragment analysis. Polymorphisms that can be identified inthis manner are termed “restriction fragment length polymorphisms”(“RFLPs”) (Glassberg, UK Patent Application 2135774; Skolnick et al.,Cytogen. Cell Genet. 32:58-67 (1982); Botstein et al., Ann. J. Hum.Genet. 32:314-331 (1980); Fischer et al., (PCT Application WO 90/13668;Uhlen, PCT Application WO 90/11369).

[0326] Polymorphisms can also be identified by Single StrandConformation Polymorphism (SSCP) analysis (Elles, Methods in MolecularMedicine: Molecular Diagnosis of Genetic Diseases, Humana Press (1996));Orita et al., Genomics 5:874-879 (1989)). A number of protocols havebeen described for SSCP including, but not limited to, Lee et al., Anal.Biochem. 205:289-293 (1992); Suzuki et al., Anal. Biochem. 192:82-84(1991); Lo et al., Nucleic Acids Research 20:1005-1009 (1992); Sarkar etal., Genomics 13:441-443 (1992). It is understood that one or more ofthe nucleic acids of the invention, may be utilized as markers or probesto detect polymorphisms by SSCP analysis.

[0327] Polymorphisms may also be found using a DNA fingerprintingtechnique called amplified fragment length polymorphism (AFLP), which isbased on the selective PCR amplification of restriction fragments from atotal digest of genomic DNA to profile that DNA (Vos et al., NucleicAcids Res. 23:4407-4414 (1995)). This method allows for the specificco-amplification of high numbers of restriction fragments, which can bevisualized by PCR without knowledge of the nucleic acid sequence. It isunderstood that one or more of the nucleic acids of the invention may beutilized as markers or probes to detect polymorphisms by AFLP analysisor for fingerprinting RNA.

[0328] Polymorphisms may also be found using random amplifiedpolymorphic DNA (RAPD) (Williams et al., Nuc. Acids Res. 18:6531-6535(1990)) and cleavable amplified polymorphic sequences (CAPS) (Lyamichevet al., Science 260:778-783 (1993)). It is understood that one or moreof the nucleic acid molecules of the invention, may be utilized asmarkers or probes to detect polymorphisms by RAPD or CAPS analysis.

[0329] Single Nucleotide Polymorphisms (SNPs) generally occur at greaterfrequency than other polymorphic markers and are spaced with a greateruniformity throughout a genome than other reported forms ofpolymorphism. The greater frequency and uniformity of SNPs means thatthere is greater probability that such a polymorphism will be found nearor in a genetic locus of interest than would be the case for otherpolymorphisms. SNPs are located in protein-coding regions and noncodingregions of a genome. Some of these SNPs may result in defective orvariant protein expression (e.g., as a result of mutations or defectivesplicing). Analysis (genotyping) of characterized SNPs can require onlya plus/minus assay rather than a lengthy measurement, permitting easierautomation.

[0330] SNPs can be characterized using any of a variety of methods. Suchmethods include the direct or indirect sequencing of the site, the useof restriction enzymes (Botstein et al., Am. J. Hum. Genet. 32:314-331(1980); Konieczny and Ausubel, Plant J. 4:403-410 (1993)), enzymatic andchemical mismatch assays (Myers et al., Nature 313:495-498 (1985)),allele-specific PCR (Newton et al., Nucl. Acids Res. 17:2503-2516(1989); Wu et al., Proc. Natl. Acad. Sci. USA 86:2757-2760 (1989)),ligase chain reaction (Barany, Proc. Natl. Acad. Sci. USA 88:189-193(1991)), single-strand conformation polymorphism analysis (Labrune etal., Am. J. Hum. Genet. 48: 1115-1120 (1991)), single base primerextension (Kuppuswarny et al., Proc. Natl. Acad. Sci. USA 88:1143-1147(1991)), Goelet U.S. Pat. No. 6,004,744; Goelet U.S. Pat. No.5,888,819), solid-phase ELISA-based oligonucleotide ligation assays(Nikiforov et al., Nucl. Acids Res. 22:4167-4175 (1994), dideoxyfingerprinting (Sarkar et al., Genomics 13:441-443 (1992)),oligonucleotide fluorescence-quenching assays (Livak et al., PCR MethodsAppl. 4:357-362 (1995a)), 5′-nuclease allele-specific hybridizationTaqMan™ assay (Livak et al., Nature Genet. 9:341-342 (1995)),template-directed dye-terminator incorporation (TDI) assay (Chen andKwok, Nucl. Acids Res. 25:347-353 (1997)), allele-specific molecularbeacon assay (Tyagi et al., Nature Biotech. 16: 49-53 (1998)), PinPointassay (Haff and Smirnov, Genome Res. 7: 378-388 (1997)), dCAPS analysis(Neff et al., Plant J. 14:387-392 (1998)), pyrose-quencing (Ronaghi etal, Analytical Biochemistry 267:65-71 (1999); Ronaghi et al PCTapplication WO 98/13523; Nyren et al PCT application WO 98/28440;www.pyrosequencing.com), using mass spectrometry, e.g. the Masscode™system (Howbert et al PCT application, WO 99/05319; Howbert et al PCTapplication WO 97/27331; www.rapigene.com; Becker et al PCT applicationWO 98/26095; Becker et al PCT application; WO 98/12355; Becker et al PCTapplication WO 97/33000; Monforte et al U.S. Pat. No. 5,965,363),invasive cleavage of oligonucleotide probes (Lyamichev et al NatureBiotechnology 17:292-296; www.twt.com), and using high densityoligonucleotide arrays (Hacia et al Nature Genetics 22:164-167;www.affymetrix.com).

[0331] Polymorphisms may also be detected using allele-specificoligonucleotides (ASO), which, can be for example, used in combinationwith hybridization based technology including southern, northern, anddot blot hybridizations, reverse dot blot hybridizations andhybridizations performed on microarray and related technology.

[0332] The stringency of hybridization for polymorphism detection ishighly dependent upon a variety of factors, including length of theallele-specific oligonucleotide, sequence composition, degree ofcomplementarity (i.e. presence or absence of base mismatches),concentration of salts and other factors such as formamide, andtemperature. These factors are important both during the hybridizationitself and during subsequent washes performed to remove targetpolynucleotide that is not specifically hybridized. In practice, theconditions of the final, most stringent wash are most critical. Inaddition, the amount of target polynucleotide that is able to hybridizeto the allele-specific oligonucleotide is also governed by such factorsas the concentration of both the ASO and the target polynucleotide, thepresence and concentration of factors that act to “tie up” watermolecules, so as to effectively concentrate the reagents (e.g., PEG,dextran, dextran sulfate, etc.), whether the nucleic acids areimmobilized or in solution, and the duration of hybridization andwashing steps.

[0333] Hybridizations are preferably performed below the meltingtemperature (T_(m)) of the ASO. The closer the hybridization and/orwashing step is to the T_(m), the higher the stringency. T_(m) for anoligonucleotide may be approximated, for example, according to thefollowing formula: T_(m)=81.5+16.6× (log 10[Na+])+0.41× 5(%G+C)−675/n;where [Na+] is the molar salt concentration of Na+ or any other suitablecation and n=number of bases in the oligonucleotide. Other formulas forapproximating T_(m) are available and are known to those of ordinaryskill in the art.

[0334] Stringency is preferably adjusted so as to allow a given ASO todifferentially hybridize to a target polynucleotide of the correctallele and a target polynucleotide of the incorrect allele. Preferably,there will be at least a two-fold differential between the signalproduced by the ASO hybridizing to a target polynucleotide of thecorrect allele and the level of the signal produced by the ASOcross-hybridizing to a target polynucleotide of the incorrect allele(e.g., an ASO specific for a mutant allele cross-hybridizing to awild-type allele). In more preferred embodiments of the presentinvention, there is at least a five-fold signal differential. In highlypreferred embodiments of the present invention, there is at least anorder of magnitude signal differential between the ASO hybridizing to atarget polynucleotide of the correct allele and the level of the signalproduced by the ASO cross-hybridizing to a target polynucleotide of theincorrect allele.

[0335] While certain methods for detecting polymorphisms are describedherein, other detection methodologies may be utilized. For example,additional methodologies are known and set forth, in Birren et al.,Genome Analysis, 4:135-186, A Laboratory Manual. Mapping Genomes, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1999); Maligaet al., Methods in Plant Molecular Biology. A Laboratory Course Manual,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1995);Paterson, Biotechnology Intelligence Unit: Genome Mapping in Plants, R.G. Landes Co., Georgetown, Tex., and Academic Press, San Diego, Calif.(1996); The Corn Handbook, Freeling and Walbot, eds., Springer-Verlag,New York, N.Y. (1994); Methods in Molecular Medicine: MolecularDiagnosis of Genetic Diseases, Elles, ed., Humana Press, Totowa, N.J.(1996); Clark, ed., Plant Molecular Biology: A Laboratory Manual, Clark,ed., Springer-Verlag, Berlin, Germany (1997).

[0336] Factors for marker-assisted selection in a plant breeding programare: (1) the marker(s) should co-segregate or be closely linked with thedesired trait; (2) an efficient means of screening large populations forthe molecular marker(s) should be available; and (3) the screeningtechnique should have high reproducibility across laboratories andpreferably be economical to use and be user-friendly.

[0337] The genetic linkage of marker molecules can be established by agene mapping model such as, without limitation, the flanking markermodel reported by Lander and Botstein, Genetics 121:185-199 (1989) andthe interval mapping, based on maximum likelihood methods described byLander and Botstein, Genetics 121:185-199 (1989) and implemented in thesoftware package MAPMAKER/QTL (Lincoln and Lander, Mapping GenesControlling Quantitative Traits Using MAPMAKER/QTL, Whitehead Institutefor Biomedical Research, Mass., (1990). Additional software includesQgene, Version 2.23 (1996), Department of Plant Breeding and Biometry,266 Emerson Hall, Cornell University, Ithaca, N.Y.). Use of Qgenesoftware is a particularly preferred approach.

[0338] A maximum likelihood estimate (MLE) for the presence of a markeris calculated, together with an MLE assuming no QTL effect, to avoidfalse positives. A log₁₀ of an odds ratio (LOD) is then calculated as:LOD=log₁₀ (MLE for the presence of a QTL/MLE given no linked QTL).

[0339] The LOD score essentially indicates how much more likely the dataare to have arisen assuming the presence of a QTL than in its absence.The LOD threshold value for avoiding a false positive with a givenconfidence, say 95%, depends on the number of markers and the length ofthe genome. Graphs indicating LOD thresholds are set forth in Lander andBotstein, Genetics 121:185-199 (1989) and further described by Arús andMoreno-González, Plant Breeding, Hayward et al., (eds.) Chapman & Hall,London, pp. 314-331 (1993).

[0340] In a preferred embodiment of the present invention the nucleicacid marker exhibits a LOD score of greater than 2.0, more preferably2.5, even more preferably greater than 3.0 or 4.0 with the trait orphenotype of interest. In a preferred embodiment, the trait of interestis altered tocopherol levels or compositions or altered tocotrienollevels or compositions.

[0341] Additional models can be used. Many modifications and alternativeapproaches to interval mapping have been reported, including the usenon-parametric methods (Kruglyak and Lander, Genetics 139:1421-1428(1995)). Multiple regression methods or models can be also be used, inwhich the trait is regressed on a large number of markers (Jansen,Biometrics in Plant Breeding, van Oijen and Jansen (eds.), Proceedingsof the Ninth Meeting of the Eucarpia Section Biometrics in PlantBreeding, The Netherlands, pp. 116-124 (1994); Weber and Wricke,Advances in Plant Breeding, Blackwell, Berlin, 16 (1994)). Procedurescombining interval mapping with regression analysis, whereby thephenotype is regressed onto a single putative QTL at a given markerinterval and at the same time onto a number of markers that serve as‘cofactors,’ have been reported by Jansen and Stam, Genetics136:1447-1455 (1994), and Zeng, Genetics 136:1457-1468 (1994).Generally, the use of cofactors reduces the bias and sampling error ofthe estimated QTL positions (Utz and Melchinger, Biometrics in PlantBreeding, van Oijen and Jansen (eds.) Proceedings of the Ninth Meetingof the Eucarpia Section Biometrics in Plant Breeding, The Netherlands,pp. 195-204 (1994), thereby improving the precision and efficiency ofQTL mapping (Zeng, Genetics 136:1457-1468 (1994)). These models can beextended to multi-environment experiments to analyzegenotype-environment interactions (Jansen et al., Theo. Appl. Genet.91:33-37 (1995)).

[0342] It is understood that one or more of the nucleic acid moleculesof the invention may be used as molecular markers. It is also understoodthat one or more of the protein molecules of the invention may be usedas molecular markers.

[0343] In a preferred embodiment, the polymorphism is present andscreened for in a mapping population, e.g. a collection of plantscapable of being used with markers such as polymorphic markers to mapgenetic position of traits. The choice of appropriate mapping populationoften depends on the type of marker systems employed (Tanksley et al.,J.P. Gustafson and R. Appels (eds.). Plenum Press, New York, pp. 157-173(1988)). Consideration must be given to the source of parents (adaptedvs. exotic) used in the mapping population. Chromosome pairing andrecombination rates can be severely disturbed (suppressed) in widecrosses (adapted x exotic) and generally yield greatly reduced linkagedistances. Wide crosses will usually provide segregating populationswith a relatively large number of polymorphisms when compared to progenyin a narrow cross (adapted x adapted).

[0344] An F₂ population is the first generation of selfing(self-pollinating) after the hybrid seed is produced. Usually a singleF₁ plant is selfed to generate a population segregating for all thegenes in Mendelian (1:2:1) pattern. Maximum genetic information isobtained from a completely classified F₂ population using a codominantmarker system (Mather, Measurement of Linkage in Heredity: Methuen andCo., (1938)). In the case of dominant markers, progeny tests (e.g., F₃,BCF₂) are required to identify the heterozygotes, in order to classifythe population. However, this procedure is often prohibitive because ofthe cost and time involved in progeny testing. Progeny testing of F₂individuals is often used in map construction where phenotypes do notconsistently reflect genotype (e.g. disease resistance) or where traitexpression is controlled by a QTL. Segregation data from progeny testpopulations e.g. F₃ or BCF₂) can be used in map construction.Marker-assisted selection can then be applied to cross progeny based onmarker-trait map associations (F₂, F₃), where linkage groups have notbeen completely disassociated by recombination events (i.e., maximumdisequilibrium).

[0345] Recombinant inbred lines (RIL) (genetically related lines;usually >F₅, developed from continuously selfing F₂ lines towardshomozygosity) can be used as a mapping population. Information obtainedfrom dominant markers can be maximized by using RIL because all loci arehomozygous or nearly so. Under conditions of tight linkage (i.e., about<10% recombination), dominant and co-dominant markers evaluated in RILpopulations provide more information per individual than either markertype in backcross populations (Reiter. Proc. Natl. Acad. Sci. (U.S.A.)89:1477-1481 (1992)). However, as the distance between markers becomeslarger (i.e., loci become more independent), the information in RILpopulations decreases dramatically when compared to codominant markers.

[0346] Backcross populations (e.g., generated from a cross between asuccessful variety (recurrent parent) and another variety (donor parent)carrying a trait not present in the former) can be utilized as a mappingpopulation. A series of backcrosses to the recurrent parent can be madeto recover most of its desirable traits. Thus a population is createdconsisting of individuals nearly like the recurrent parent but eachindividual carries varying amounts or mosaic of genomic regions from thedonor parent. Backcross populations can be useful for mapping dominantmarkers if all loci in the recurrent parent are homozygous and the donorand recurrent parent have contrasting polymorphic marker alleles (Reiteret al., Proc. Natl. Acad. Sci. (U.S.A.) 89:1477-1481 (1992)).Information obtained from backcross populations using either codominantor dominant markers is less than that obtained from F₂ populationsbecause one, rather than two, recombinant gamete is sampled per plant.Backcross populations, however, are more informative (at low markersaturation) when compared to RILs as the distance between linked lociincreases in RIL populations (i.e. about 0.15% recombination). Increasedrecombination can be beneficial for resolution of tight linkages, butmay be undesirable in the construction of maps with low markersaturation.

[0347] Near-isogenic lines (NIL) (created by many backcrosses to producea collection of individuals that is nearly identical in geneticcomposition except for the trait or genomic region under interrogation)can be used as a mapping population. In mapping with NILs, only aportion of the polymorphic loci is expected to map to a selected region.

[0348] Bulk segregant analysis (BSA) is a method developed for the rapididentification of linkage between markers and traits of interest(Michelmore et al., Proc. Natl. Acad. Sci. U.S.A. 88:9828-9832 (1991)).In BSA, two bulked DNA samples are drawn from a segregating populationoriginating from a single cross. These bulks contain individuals thatare identical for a particular trait (resistant or susceptible toparticular disease) or genomic region but arbitrary at unlinked regions(i.e. heterozygous). Regions unlinked to the target region will notdiffer between the bulked samples of many individuals in BSA.

[0349] In an aspect of the present invention, one or more of the nucleicmolecules of the present invention are used to determine the level(i.e., the concentration of mRNA in a sample, etc.) in a plant(preferably canola, corn, Brassica campestris, oilseed rape, rapeseed,soybean, crambe, mustard, castor bean, peanut, sesame, cottonseed,linseed, safflower, oil palm, flax or sunflower) or pattern (i.e., thekinetics of expression, rate of decomposition, stability profile, etc.)of the expression of a protein encoded in part or whole by one or moreof the nucleic acid molecule of the present invention (collectively, the“Expression Response” of a cell or tissue).

[0350] As used herein, the Expression Response manifested by a cell ortissue is said to be “altered” if it differs from the ExpressionResponse of cells or tissues of plants not exhibiting the phenotype. Todetermine whether an Expression Response is altered, the ExpressionResponse manifested by the cell or tissue of the plant exhibiting thephenotype is compared with that of a similar cell or tissue sample of aplant not exhibiting the phenotype. As will be appreciated, it is notnecessary to re-determine the Expression Response of the cell or tissuesample of plants not exhibiting the phenotype each time such acomparison is made; rather, the Expression Response of a particularplant may be compared with previously obtained values of normal plants.As used herein, the phenotype of the organism is any of one or morecharacteristics of an organism (e.g. disease resistance, pest tolerance,environmental tolerance such as tolerance to abiotic stress, malesterility, quality improvement or yield etc.). A change in genotype orphenotype may be transient or permanent. Also as used herein, a tissuesample is any sample that comprises more than one cell. In a preferredaspect, a tissue sample comprises cells that share a commoncharacteristic (e.g. Derived from root, seed, flower, leaf, stem orpollen etc.).

[0351] In one aspect of the present invention, an evaluation can beconducted to determine whether a particular mRNA molecule is present.One or more of the nucleic acid molecules of the present invention areutilized to detect the presence or quantity of the mRNA species. Suchmolecules are then incubated with cell or tissue extracts of a plantunder conditions sufficient to permit nucleic acid hybridization. Thedetection of double-stranded probe-mRNA hybrid molecules is indicativeof the presence of the mRNA; the amount of such hybrid formed isproportional to the amount of mRNA. Thus, such probes may be used toascertain the level and extent of the mRNA production in a plant's cellsor tissues. Such nucleic acid hybridization may be conducted underquantitative conditions (thereby providing a numerical value of theamount of the mRNA present). Alternatively, the assay may be conductedas a qualitative assay that indicates either that the mRNA is present,or that its level exceeds a user set, predefined value.

[0352] A number of methods can be used to compare the expressionresponse between two or more samples of cells or tissue. These methodsinclude hybridization assays, such as northerns, RNAse protectionassays, and in situ hybridization. Alternatively, the methods includePCR-type assays. In a preferred method, the expression response iscompared by hybridizing nucleic acids from the two or more samples to anarray of nucleic acids. The array contains a plurality of suspectedsequences known or suspected of being present in the cells or tissue ofthe samples.

[0353] An advantage of in situ hybridization over more conventionaltechniques for the detection of nucleic acids is that it allows aninvestigator to determine the precise spatial population (Angerer etal., Dev. Biol. 101:477-484 (1984); Angerer et al., Dev. Biol.112:157-166 (1985); Dixon et al., EMBO J. 10:1317-1324 (1991)). In situhybridization may be used to measure the steady-state level of RNAaccumulation (Hardin et al., J. Mol. Biol. 202:417-431 (1989)). A numberof protocols have been devised for in situ hybridization, each withtissue preparation, hybridization and washing conditions (Meyerowitz,Plant Mol. Biol. Rep. 5:242-250 (1987); Cox and Goldberg, In: PlantMolecular Biology: A Practical Approach, Shaw (ed.), pp. 1-35, IRLPress, Oxford (1988); Raikhel et al., In situ RNA hybridization in planttissues, In: Plant Molecular Biology Manual, vol. B9:1-32, KluwerAcademic Publisher, Dordrecht, Belgium (1989)).

[0354] In situ hybridization also allows for the localization ofproteins within a tissue or cell (Wilkinson, In Situ Hybridization,Oxford University Press, Oxford (1992); Langdale, In Situ HybridizationIn: The Corn Handbook, Freeling and Walbot (eds.), pp. 165-179,Springer-Verlag, New York (1994)). It is understood that one or more ofthe molecules of the invention, preferably one or more of the nucleicacid molecules or fragments thereof of the invention or one or more ofthe antibodies of the invention may be utilized to detect the level orpattern of a protein or mRNA thereof by in situ hybridization.

[0355] Fluorescent in situ hybridization allows the localization of aparticular DNA sequence along a chromosome, which is useful, among otheruses, for gene mapping, following chromosomes in hybrid lines, ordetecting chromosomes with translocations, transversions or deletions.In situ hybridization has been used to identify chromosomes in severalplant species (Griffor et al., Plant Mol. Biol. 17:101-109 (1991);Gustafson et al., Proc. Natl. Acad. Sci. (U.S.A.) 87:1899-1902 (1990);Mukai and Gill, Genome 34:448-452 (1991); Schwarzacher andHeslop-Harrison, Genome 34:317-323 (1991); Wang et al., Jpn. J. Genet.66:313-316 (1991); Parra and Windle, Nature Genetics 5:17-21 (1993)). Itis understood that the nucleic acid molecules of the invention may beused as probes or markers to localize sequences along a chromosome.

[0356] Another method to localize the expression of a molecule is tissueprinting. Tissue printing provides a way to screen, at the same time onthe same membrane many tissue sections from different plants ordifferent developmental stages (Yomo and Taylor, Planta 112:35-43(1973); Harris and Chrispeels, Plant Physiol. 56:292-299 (1975); Cassaband Varner, J. Cell. Biol. 105:2581-2588 (1987); Spruce et al.,Phytochemistry 26:2901-2903 (1987); Barres et al., Neuron 5:527-544(1990); Reid and Pont-Lezica, Tissue Printing: Tools for the Study ofAnatomy, Histochemistry and Gene Expression, Academic Press, New York,N.Y. (1992); Reid et al., Plant Physiol. 93:160-165 (1990); Ye et al.,Plant J. 1:175-183 (1991)).

[0357] One skilled in the art can refer to general reference texts fordetailed descriptions of known techniques discussed herein or equivalenttechniques. These texts include Current Protocols in Molecular BiologyAusubel, et al., eds., John Wiley & Sons, N.Y. (1989), and supplementsthrough September (1998), Molecular Cloning, A Laboratory Manual,Sambrook et al, 2^(nd) Ed., Cold Spring Harbor Press, Cold SpringHarbor, N.Y. (1989), Genome Analysis: A Laboratory Manual 1: AnalyzingDNA, Birren et al., Cold Spring Harbor Press, Cold Spring Harbor, N.Y.(1997); Genome Analysis: A Laboratory Manual 2: Detecting Genes, Birrenet al., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1998);Genome Analysis: A Laboratory Manual 3: Cloning Systems, Birren et al.,Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1999); GenomeAnalysis: A Laboratory Manual 4: Mapping Genomes, Birren et al., ColdSpring Harbor Press, Cold Spring Harbor, N.Y. (1999); Plant MolecularBiology: A Laboratory Manual, Clark, Springer-Verlag, Berlin, (1997),Methods in Plant Molecular Biology, Maliga et al., Cold Spring HarborPress, Cold Spring Harbor, N.Y. (1995). These texts can, of course, alsobe referred to in making or using an aspect of the invention. It isunderstood that any of the agents of the invention can be substantiallypurified and/or be biologically active and/or recombinant.

[0358] Having now generally described the invention, the same will bemore readily understood through reference to the following examples thatare provided by way of illustration, and are not intended to be limitingof the present invention, unless specified.

EXAMPLE 1

[0359] A DNA sequence of gamma-tocopherol methyltransferase fromArabidopsis thaliana (NCBI General Identifier Number 4106537) is used tosearch databases for plant sequences with homology to GMT using BLASTN(Altschul et al., Nucleic Acids Res. 15 25:3389-3402 (1997); see alsowww.ncbi.nlm.nih.gov/BLAST/). Results are shown in table 1, below. TABLE1 BLAST RESULTS FOR PLANT SEQUENCES ENCODING POLYPEPTIDES HOMOLOGOUS TOARABIDOPSIS GAMMA-TOCOPHEROL METHYLTRANSFERASE Sequences producingsignificant alignments: Score (bits) E Value Arabidopsis thaliana(Columbia ecotype) 707 0.0 Brassica napus S8 clone 611 e−179 Brassicanapus P4 clone 605 e−177 cotton GMT 459 e−133 soybeanGMT2 454 e−132soybeanGMT1 453 e−132 soybeanGMT3 453 e−131 Marigold GMT (Tageteserecta) 446 e−129 tomato GMT 441 e−128 cuphea GMT 440 e−127 Rice GMT 430e−124 corn GMT 428 e−123 sorghum bicolor GMT 328 9e−94 

[0360] The protein identity of these sequences compared to one anotheris listed in table 2. TABLE 2 PROTEIN IDENTITY TABLE OF PLANT SEQUENCESENCODING POLYPEPTIDES HOMOLOGOUS TO GAMMA-TOCOPHEROL METHYLTRANSFERASEArabidopsis GMT Arabidopsis Brassica Brassica Cuphea Gossypium Zea OryzaSorghum Tagetes (gi 4106537) Columbia S8 P4 pulcherrima hirsutum mayssativa bicolor erecta Arabidopsis GMT 100%  (gi 4106537) 348/348Arabidopsis 99% 100%  Columbia GMT 347/348 348/348 Brassica S8 GMT 88%88% 100%  309/350 308/350 347/347 Brassica P4 GMT 87% 86% 96% 100% 304/349 303/349 335/348 347/347 Cuphea pulcherrima 72% 71% 68% 68% 100% GMT 213/295 212/295 216/314 213/313 376/376 Gossypium hirsutum GMT 67%67% 71% 67% 71% 100%  218/323 219/323 225/316 231/342 212/296 345/345Zea mays GMT 63% 62% 65% 63% 71% 67% 100%  210/333 209/333 217/332211/330 208/290 223/331 352/352 Oryza sativa GMT 63% 63% 67% 62% 70% 65%76% 100%  212/332 212/332 214/319 220/352 204/291 226/347 279/364364/364 Sorghum bicolor GMT 72% 72% 75% 73% 74% 78% 96% 91% 100% 154/212 153/212 159/212 156/212 157/212 166/212 208/215 193/212 215/215Tagetes erecta GMT 69% 70% 69% 68% 72% 70% 70% 71% 77% 100% 218/312219/312 214/309 211/310 210/291 209/297 216/305 219/308 165/212 310/310Lycopersicon 68% esculentum GMT 212/311 Glycine max GMT1 73% 218/297Glycine max GMT2 70% 225/318 Glycine max GMT3 75% 220/290

[0361] A protein sequence of the Synechocystis GMT (NCBI GeneralIdentifier Number 1001725) is used in a BlastP search against predictedORFs from other cyanobacteria at the ERGO website(www.integratedgenomics.com/IGwit/).

[0362] Two sequences with substantial homology to the Synechocystis GMTare found from two cyanobacteria species. These sequences are annotatedas having a function of delta(24)-sterol C-methyltransferase (EC2.1.1.41). E-Value Score Nostoc punctiforme 1e−105 375 Anabaena sp.1e−101 361

[0363] TABLE 3 CYANOBACTERIA GMT CLUSTAL W (1.8) MULTIPLE SEQUENCEALIGNMENT Nostoc punctiforme-------------------------MSATLYQQIQQFYDASSGLWEQIWGEHMHHG (SEQ ID NO: 39)Anabaena sp.-----------------------------MSATLYQQIQQFYDASSGLWEEIWGEHMHHG (SEQ ID NO:40) SynechocystisMVYHVRPKHALFLAFYCYFSLLTMASATIASADLYEKIKNFYDDSSGLWEDVWGEHMHHG (SEQ ID NO:41)                                                  ** **::*::*********::******** Nostoc punctiformeYYGADGTQKKDRRQAQIDLIEELLNWAGVQAAED---LDVGCGIGGSSLYTLAQKFNAKA Anabaenasp. YYGADGTEQKNRRQAQIDLIEELLTWAGVQTAEN---LDVGCGIGGSSLYLAGKLNAKASynechocystisYGPHGTYRIDRRQAQIDLIKELLAWAVPQNSAKPRKILDLGCGIGGSSLYLAQQNQAEV                    ***..** : :*********:*** **  * : .   ***:************ :*:. Nostoc punctiformeGITLSPVQAARATERALEANLSLRTQFQVANAQAMPFADDSFDLVWSLESGEHMPDKTK Anabaena sp.GITLSPVQAARATERAKEAGLSGRSQFLVANAQAMPFDDNSFDLVWSLESGEHMPDKTKSynechocystisMGASLSPVQVERAGERARALGLGSTCQFQVANALDLPFASDSFDWVWSLESGEHMPNKAQ                     * :*****. ** ***   .*.   ** ****  :** .:*************:*:: Nostoc punctiformeFLQECYRVLKPGGKLIMVTWCHRPTD--ESPLTADEEKHLQDIYRVYCLPYVISLPEYEA Anabaenasp. FLQECYRVLKPGGKLIMVTWCHRPTD--KTPLTADEKKHLEDIYRVYCLPYVISLPEYEASynechocystisFLQEAWRVLKPGGRLILATWCHRPIDPGNGPLTADERRHLQAIYDVYCLPYVVSLPDYEA                   ****.:*******:**:.****** *  : ******.:**: *********:***:*** Nostoc punctiformeIAHQLPLHNIRTADWSTAVAPFWNVVIDSAFTPQALWGLLNAGWTTIQGALSLGLMRRGY Anabaenasp. IARQLPLNNIRTADWSQSVAQFWNIVIDSAFTPQAIFGLLRAGWTTIQGALSLGLMRRGYSynechocystisIARECGFGEIKTADWSVAVAPFWDRVIESAFDPRVLWALGQAGPKIINAALCLRLMKWGY                   **::  : :*:***** :** **: **:*** *:.::.* .** . *:.**.* **:** Nostoc punctiforme ERGLIRFGLLCGNK--- Anabaena sp. ERGLIRFGLLCGDK---Synechocystis ERGLVRFGLLTGIKPLV ****:***** * *

[0364] The sequence of the Synechocystis MT1 (NCBI General IdentifierNumber 1653572) is used in a blast search against ESTs of othercyanobacteria at the ERGO website (www.integratedgenomics.com/IGwit/).

[0365] Three sequences with substantial homology to the SynechocystisMT1 are found from three cyanobacteria species. These sequences are allannotated as having a function of DELTA(24)-STEROL C-METHYLTRANSFERASE(EC 2.1.1.41) BlastP SCORE Anabaena sp. 1e−144 504 Synechococcus sp.6e−98  350 Prochlorococcus marinus 2e−84  304

[0366] TABLE 4 CYANOBACTERIA MT1 CLUSTAL W (1.8) MULTIPLE SEQUENCEALIGNMENT SynechocystisMPEYLLLPAGLISLSLAIAAGLYLLTARGYQSSDSVANAYDQWTEDGILEYYWGDHIHLG (SEQ ID NO:46) Anabaena-MSWLFSTLVFFLTLLTAGIALYLITARRYQSSNSVANSYDQWTEDGILEFYWGEHIHLG (SEQ ID NO:47) Synechococcus---MLAGLLLLTGAAGATALLIWLQRDRRYHSSDSVAAAYDAWTDDQLLERLWGDHVHLG (SEQ ID NO:48) ProchlorococcusMSIFLISSLVIFLTLLFSSLILWRINTRKYISSRTVATAYDSWTQDKLLERLWGEHIHLG (SEQ ID NO:49)     *     :       .  ::    * * ** :** :** **:* :**  **:*:***SynechocystisHYGDPPVAKDFIQSKIDFVHAMAQWGGLDTLPPGTTVLDVGCGIGGSSRILAKDYGFNVT AnabaenaHYGSPPQRKDFLVAKSDFVHEMVRWGGLDKLPPGTTLLDVGCGIGGSSRILARDYGFAVTSynechococcusHYGNPPGSVDFRQAKEAFVHELVRWSGLDQLPRGSRVLDVGCGIGGSARILARDYGLDVLProchlorococcusFYP-LNKNIDFREAKVQFVHELVSWSGLDKLPRGSRILDVGCGIGGSSRILANYYGFNVT.*       **  :*  *** :. *.*** ** *: :**********:****. **: *SynechocystisGITISPQQVKRATELTPPDVTAKFAVDDAMALSFPDGSFDVVWSVEAGPHMPDKAVFAKE AnabaenaGITISPQQVQRAQELTPQELNAQFLVDDAMALSFPDNSFDVVWSIEAGPHMPDKAIFAKESynechococcusGVSISPAQIRRATELTPAGLSCRFEVMDALNLQLPDRQFDAVWTVEAGPHMPDKQRFADEProchlorococcusGITISPAQVKRAKELTPYECKCNFKVMDALDLKFEEGIFDGVWSVEAGAHMNNKTKFADQ*::*** *::** ****   ...* * **: *.: :  ** **::***.** :*  **.:SynechocystisLLRVVKPGGILVVADWNQRDDRQVPLNFWEKPVMRQLLDQWSHPAFASIEGFAENLEATG AnabaenaLMRVLKPGGIMVLADWNQRDDRQKPLNFWEKPVMQQLLDQWSHPAFSSIEGFSELLAATGSynechococcusLLRVLRPGGCLAAADWNRRAPKDGAMNSTERWVMRQLLNQWAHPEFASISGFRANLEASPProchlorococcusMLRTLRPGGYLALADWNSRDLQKQPPSMIEKIILKQLLEQWVHPKFISINEFSSILINNK::*.::*** :. **** *  :. . .  *: :::***:** ** * **. *   *  .SynechocystisLVEGQVTTADWTVPTLPAWLDTIWQGIIRPQGWLQYGIRGFIKSVREVPTILLMRLAFGV AnabaenaLVEGEVITADWTKQTLPSWLDSIWQGIVRPEGLVRFGLSGFIKSLREVPTLLLMRLAFGTSynechococcusHQRGLISTGDWTLATLPSWFDSIAEGLRRPWAVLGLGPKAVLQGLRETPTLLLMHWAFATProchlorococcusNSSGQVISSNWNSFTNPSWFDSIFEGMRRPNSILSLGPGAIIKSIREIPTILLMDWAFKK   * : :.:*.  * *:*:*:* :*: ** . :  *  ..::.:** **:***  ** SynechocystisGLCRFGMFKAVRKNATQA------------- Anabaena GLCRFGMFRALRADTVRSSAEQTSAIKVAQKSynechococcus GLMQFGVFRLSR------------------- ProchlorococcusGLMEFGVYKCRG------------------- ** .**:::

EXAMPLE 2

[0367] Constructs are prepared to direct expression of the Arabidopsis,P4 and S8 Brassica napus, Cuphea pulcherrima, and Gossypium hirsutum GMTsequences in plants. The coding region of each GMT is amplified fromeither the appropriate EST clone or cDNA, as appropriate. Doublestranded DNA sequence is obtained of all PCR products to verify that noerrors are introduced by the PCR amplification.

[0368] An S8 Brassica GMT coding sequence is amplified from Brassicanapus leaf cDNA as follows: PolyA⁺ RNA is isolated from Brassica napus(var. Quantum) leaf tissue using an adapted biotin/streptavadinprocedure based on the “mRNA Capture Kit” by Roche MolecularBiochemicals (Indianapolis, Ind.). Young leaf tissue is homogenized inCTAB buffer (50 mM Tris-HCl pH 9, 0.8M NaCl, 0.5% CTAB, 10 mM EDTA),extracted with chloroform, and pelleted. As specified by themanufacturer's instructions, polyA⁺ RNA in the soluble fraction ishybridized to biotin-labeled oligo-dT, immobilized onstreptavadin-coated PCR tubes and washed. First strand cDNA issynthesized using the “1^(st) strand cDNA synthesis kit for RT-PCR”(Roche Molecular Biochemicals) in a 50 μl volume according to themanufacturer's protocol. Following the cDNA synthesis, the solublecontents of the tube are replaced with equal volume amplificationreaction mixture. Components of the mixture at final concentrationconsisted of: 1×Buffer 2 (Expand™ High Fidelity PCR System, RocheMolecular Biochemicals), 200 μM dNTPs, 0.5 units RNAseH, 300 nM eachsynthetic oligonucleotide primers #16879 (SEQ ID NO: 51) and #16880 (SEQID NO: 52) and 0.4 μl Expand™ High Fidelity Polymerase (Roche MolecularBiochemicals).

[0369] A GMT gene is PCR amplified for 30 cycles using a “touchdown”cycling profile: 15 min pre-incubation at 37° C. followed by a 3 minpre-incubation at 94° C., during which Expand™ polymerase is spiked intothe mix. The product is then amplified for 15 cycles consisting ofdenaturation at 94° C. for 30 sec, annealing at 65° C. for 30 sec, andelongation at 72° C. for 1.5 min. The annealing temperature is decreasedby 1° C. per cycle for each of the previous 15 cycles. An additional 15cycles followed, consisting of 94° C. for 30 sec, 50° C. for 30 sec, and72° C. for 1.5 min, followed by a 7 min hold 72° C.

[0370] The resulting PCR product is desalted using the Pharmacia“MicroSpin™ S-400 HR Column” (Pharmacia, Uppsala, Sweden) then clonedinto the vector pCR2.1 using the “TOPO TA Cloning® Kit” (Invitrogen,Carlsbad, Calif.) according to manufacturer's instructions. Theresultant intermediate plasmid is named pMON67178 and confirmed by DNAsequencing. A pMON67178 intermediate plasmid is digested with therestriction endonucleases NotI and Sse8387I to liberate a S8 BrassicaGMT insert, which is then gel-purified using the “QIAquick GelExtraction Kit” (QIAGEN Inc., Valencia, Calif.). The vector pCGN9979(FIG. 2) is prepared by digesting with NotI and Sse8387I endonucleases.Enzymes are subsequently removed using “StrataClean Resin™” (Stratagene,La Jolla, Calif.) followed by “MicroSpin™ S-400 HR Column” treatment(Pharmacia, Uppsala, Sweden). A GMT insert is ligated into the pCGN9979vector, resulting in the formation of the binary construct pMON67170.

[0371] An Arabidopsis GMT coding sequence is amplified from Arabidopsisthaliana, ecotype Columbia using the same methodology as described abovefor the S8 Brassica GMT with the exceptions that RNAseH is not added tothe amplification reaction mixture, and the synthetic oligonucleotideprimers are #16562 (SEQ ID NO: 75) and #16563 (SEQ ID NO: 76). Theresulting PCR product is desalted using the Pharmacia “MicroSpin™ S-400HR Column” (Pharmacia, Uppsala, Sweden) then cloned into the vectorpCR2.1 using the “TOPO TA Cloning® Kit” (Invitrogen, Carlsbad, Calif.)according to manufacturer's instructions. The resultant intermediateplasmid is named pMON67155 and confirmed by DNA sequencing. ThepMON67155 intermediate plasmid is digested with the restrictionendonucleases NotI and Sse8387I to liberate an Arabidopsis thaliana GMTinsert, which is then gel-purified using the “QIAquick Gel ExtractionKit” (QIAGEN Inc., Valencia, Calif.). The vector pCGN9979 is prepared bydigesting with NotI and Sse8387I endonucleases. Enzymes are subsequentlyremoved using “StrataClean Resin™” (Stratagene, La Jolla, Calif.)followed by “MicroSpin™ S-400 HR Column” treatment (Pharmacia, Uppsala,Sweden). A GMT insert is ligated into the pCGN9979 vector, resulting inthe formation of the binary construct pMON67156.

[0372] A P4 Brassica GMT coding sequence is amplified from Brassicanapus leaf cDNA using the same methodology as described above for the S8Brassica GMT with the exceptions that RNAseH is not added to theamplification reaction mixture, and the synthetic oligonucleotideprimers are #16655 (SEQ ID NO: 53) and #16654 (SEQ ID NO: 54). A“touchdown” cycling conditions consisted of a pre-incubation for 3 minat 94° C., during which 0.4 μl Expand polymerase is spiked into the mix.The product is then amplified with 15 cycles of denaturation at 94° C.for 30 sec, annealing at 60° C. for 30 sec, and elongation at 72° C. for1.5 min. The annealing temperature is decreased by 1° C. per cycle foreach of the previous 15 cycles. An additional 15 cycles followed,consisting of 94° C. for 30 sec, 45° C. for 30 sec, and 72° C. for 1.5min, followed by a 7 min hold at 72° C.

[0373] The resulting PCR product is desalted using the Pharmacia“MicroSpin™ S-400 HR Column” (Pharmacia, Uppsala, Sweden) then clonedinto the GATEWAY vector pDONR™ 201 using the “PCR Cloning System withGATEWAY Technology” (Life Technologies, a Division of InvitrogenCorporation, Rockville, Md.), according to the manufacturer'sinstructions. The ensuing plasmid pMON68751 is confirmed by DNAsequencing.

[0374] A P4 Brassica GMT is then cloned from the pMON68751 donor vectorinto the pMON67150 destination vector, which is the GATEWAY-compatibleversion of the pCGN9979 Napin binary. The “E. coli Expression Systemswith GATEWAY Technology” kit (Life Technologies, a Division ofInvitrogen Corporation, Rockville, Md.) is used according to themanufacturer's instructions to create the expression clone pMON67159.

[0375] A Cuphea pulcherrima GMT coding sequence is amplified from theEST clone LIB3792-031-Q1-K1-F3 using the synthetic oligonucleotideprimers #16658 (SEQ ID NO: 55) and #16659 (SEQ ID NO: 56). 1.0 μl of ESTtemplate is used for the Cuphea GMT amplification reaction. Otherwise,amplification conditions and cycling parameters are identical to thoseof P4 Brassica GMT.

[0376] Using the same GATEWAY procedure as described above for the P4Brassica GMT coding region, a Cuphea GMT PCR product is cloned into thepDONR™ 201 vector to create pMON68752, then subcloned into the Napinexpression vector pMON67150 to create pMON67158.

[0377] A Gossypium hirsutum GMT coding sequence is amplified from theEST clone LIB3584-003-P1-K1-Al using the synthetic oligonucleotideprimers #16681 (SEQ ID NO: 57) and #16682 (SEQ ID NO: 58). 0.51 μl ofEST template is used for the Gossypium GMT amplification reaction.Otherwise, amplification conditions and cycling parameters are identicalto those of P4 Brassica GMT.

[0378] Using the same GATEWAY procedure as described above for the P4Brassica GMT coding region, a Gossypium GMT PCR product is cloned intothe pDONR™ 201 vector to create pMON67161, then subcloned into a napinexpression vector pMON67150 to create pMON67160.

[0379] The napin cassette derived from pCGN3223 (described in U.S. Pat.No. 5,639,790) is used to drive the expression of GMT sequences inseeds. GMT sequences are cloned into the multiple cloning site of thenapin cassette using either a NotISse8387I digest (pMON67178) or thegateway cloning system (Gibco BRL) in a binary vector suitable for planttransformation (pCGN9979).

[0380] The resulting plasmids containing the gene of interest in theplant binary transformation vector under the control of the napinpromoter are labeled as follows pMON67156 (Arabidopsis thaliana,Columbia ecotype), pMON67170 (S8 Brassica napus GMT), pMON67159 (P4Brassica napus GMT), pMON 67158 (Cuphea pulcherrima GMT), and pMON 67160(Gossypium hirsutum GMT).

[0381] The plant binary constructs described above are used inArabidopsis thaliana plant transformation to direct the expression ofthe gamma-methyltransferases in the embryo. Binary vector constructs aretransformed into ABI strain Agrobacterium cells by the method ofHolsters et al. Mol. Gen. Genet. 163:181-187 (1978). TransgenicArabidopsis thaliana plants are obtained by Agrobacterium-mediatedtransformation as described by Valverkens et al., Proc. Nat. Acad. Sci.85:5536-5540 (1988), Bent et al., Science 265:1856-1860 (1994), andBechtold et al, C.R. Acad. Sci., Life Sciences 316:1194-1199 (1993).Transgenic plants are selected by sprinkling the transformed T₁ seedsdirectly onto soil and then vernalizing them at 4° C. in the absence oflight for 4 days. The seeds are then transferred to 21° C., 16 hourslight and sprayed with a 1:200 dilution of Finale (Basta) at 7 days and14 days after seeding. Transformed plants are grown to maturity and theT₂ seed that is produced is analyzed for tocopherol content. FIGS. 21a,21 b, 22 a, and 22 b show the tocopherol analysis from T2 seed oftransgenic Arabidopsis thaliana plants expressing GMTs from differentsources under the control of the napin seed-specific promoter. FIG. 23is a graph showing average seed α-tocopherol levels for various lines oftransformed plants. In FIG. 23, the plant lines shown have the followingGMT sequence origins: 67156=Arabidopsis GMT, 67158=Cuphea GMT,67159=Brassica (P4)GMT, 67160=Cotton GMT, and 67170=Brassica (S8) GMT.Table 5 below gives specific tocopherol level results for varioustransformed and control plant lines. TABLE 5 ng α ng β ng γ ng δ ngtotal toco/mg toco/mg toco/mg toco/mg toco/mg % Avg. seed seed seed seedseed Line Number A. description Gen. Alpha Alpha % 6.64 15.28 494.9113.16 529.99  9979-36  9979 = vector control 1.3 1.3 6.07 15.69 490.8213.66 526.23  9979-37  9979 = vector control 1.2 6.57 16.79 492.59 12.37528.32  9979-38  9979 = vector control 1.2 7.76 17.16 513.41 15.76554.09  9979-39  9979 = vector control 1.4 8.44 15.62 508.64 15.94548.64  9979-40  9979 = vector control 1.5 291.45 21.86 180.41 4.96498.69 67156-8 67156 = napin GMT arab T2 58.4 75.5 275.80 20.49 141.253.05 440.59 67156-6 67156 = napin GMT arab T2 62.6 289.41 21.00 138.563.73 452.70 67156-12 67156 = napin GMT arab T2 63.9 312.57 22.56 128.322.91 466.36 67156-5 67156 = napin GMT arab T2 67.0 302.71 20.69 113.962.53 439.89 67156-3 67156 = napin GMT arab T2 68.8 329.09 24.38 118.803.37 475.65 67156-1 67156 = napin GMT arab T2 69.2 352.00 21.78 128.753.54 506.08 67156-9 67156 = napin GMT arab T2 69.6 304.60 19.54 110.642.65 437.43 67156-11 67156 = napin GMT arab T2 69.6 337.70 24.25 109.932.86 474.74 67156-15 67156 = napin GMT arab T2 71.1 359.35 20.72 39.850.31 420.23 67156-13 67156 = napin GMT arab T2 85.5 367.77 22.54 35.410.35 426.08 67156-14 67156 = napin GMT arab T2 86.3 373.10 22.67 27.930.11 423.82 67156-10 67156 = napin GMT arab T2 88.0 383.43 23.64 24.000.26 431.33 67156-2 67156 = napin GMT arab T2 88.9 385.72 22.61 10.770.00 419.10 67156-4 67156 = napin GMT arab T2 92.0 412.47 27.18 13.000.21 452.86 67156-7 67156 = napin GMT arab T2 91.1 296.50 23.38 163.937.58 491.39 67159-3 67159 = brassica P4 GMT T2 60.3 69.1 327.29 3.46192.06 9.38 532.18 67159-13 Brassica P4 GMT T2 61.5 294.64 18.61 148.426.93 468.60 67159-2 67159 = brassica P4 GMT T2 62.9 309.72 21.32 152.466.20 489.70 67159-7 67159 = brassica P4 GMT T2 63.2 300.73 21.11 142.665.67 470.18 67519-1 67159 = brassica P4 GMT T2 64.0 305.37 20.25 141.837.85 475.29 67159-10 67159 = brassica P4 GMT T2 64.2 311.90 20.92 145.606.91 485.33 67159-5 67159 = brassica P4 GMT T2 64.3 289.83 19.63 128.076.33 443.86 67159-12 67159 = brassica P4 GMT T2 65.3 302.93 17.84 127.915.36 454.03 67159-6 67159 = brassica P4 GMT T2 66.7 348.38 19.53 103.127.50 478.53 67159-9 67159 = brassica P4 GMT T2 72.8 329.10 20.27 78.654.28 432.30 67159-15 67159 = brassica P4 GMT T2 76.1 359.15 23.04 70.614.95 457.76 67159-11 67159 = brassica P4 GMT T2 78.5 358.83 19.79 68.264.79 451.67 67159-14 67159 = brassica P4 GMT T2 79.4 398.21 19.29 32.823.20 453.52 67159-4 67159 = brassica P4 GMT T2 87.8 3.97 0.00 494.6715.15 513.79  9979-81 control 0.8 0.8 3.32 0.00 501.58 18.47 523.37 9979-82 control 0.6 4.00 0.00 492.08 15.31 511.38  9979-83 control 0.84.19 0.00 541.20 18.42 563.81  9979-84 control 0.7 5.23 0.00 541.7520.12 567.10  9979-85 control 0.9 251.34 10.02 216.55 6.77 484.6867158-8 napin Cuphea GMT T2 51.9 77.3 325.52 10.51 156.76 5.32 498.1167158-11 napin Cuphea GMT T2 65.4 338.00 10.58 155.40 5.35 509.3367158-12 napin Cuphea GMT T2 66.4 322.09 8.99 139.84 4.74 475.66 67158-5napin Cuphea GMT T2 67.7 348.47 12.70 132.54 5.14 498.85 67158-10 napinCuphea GMT T2 69.9 369.43 14.85 135.94 4.49 524.71 67158-15 napin CupheaGMT T2 70.4 324.99 9.08 123.23 3.95 461.25 67158-4 napin Cuphea GMT T270.5 358.91 8.49 108.56 3.76 479.72 67158-9 napin Cuphea GMT T2 74.8363.29 14.16 84.19 3.45 465.09 67158-3 napin Cuphea GMT T2 78.1 375.189.78 46.59 2.39 433.94 67158-1 napin Cuphea GMT T2 86.5 425.61 13.1439.87 2.71 481.32 67158-13 napin Cuphea GMT T2 88.4 415.44 13.75 33.162.01 464.35 67158-7 napin Cuphea GMT T2 89.5 452.35 15.65 21.65 3.46493.10 67158-2 napin Cuphea GMT T2 91.7 430.11 20.33 9.67 0.00 460.1167158-14 napin Cuphea GMT T2 93.5 408.68 13.89 7.13 1.22 430.92 67158-6napin Cuphea GMT T2 94.8 6.18 0.00 510.97 19.47 536.62  9979-86 control1.2 0.9 4.33 0.00 547.85 21.06 573.24  9979-87 control 0.8 6.28 0.00503.21 19.67 529.17  9979-88 control 1.2 4.35 0.00 538.55 21.08 563.98 9979-89 control 0.8 3.45 0.00 523.43 19.31 546.19  9979-90 control 0.65.52 0.47 478.70 17.54 502.23 67160-7 napin cotton GMT T2 1.1 65.1 8.110.00 552.24 21.34 581.69 67160-15 napin cotton GMT T2 1.4 324.58 7.93177.97 7.70 518.18 67160-9 napin cotton GMT T2 62.6 338.02 7.43 160.279.11 514.82 67160-1 napin cotton GMT T2 65.7 345.35 9.94 159.12 7.51521.92 67160-5 napin cotton GMT T2 66.2 355.54 9.65 155.73 6.95 527.8767160-14 napin cotton GMT T2 67.4 371.70 14.34 142.80 6.58 535.4367160-2 napin cotton GMT T2 69.4 355.35 5.96 135.17 9.11 505.59 67160-11napin cotton GMT T2 70.3 360.43 7.03 136.83 7.76 512.05 67160-6 napincotton GMT T2 70.4 373.32 9.65 138.68 7.74 529.39 67160-4 napin cottonGMT T2 70.5 374.20 10.97 89.34 4.57 479.07 67160-3 napin cotton GMT T278.1 435.98 16.16 67.09 4.81 524.03 67160-8 napin cotton GMT T2 83.2446.18 13.59 44.43 3.54 507.74 67160-12 napin cotton GMT T2 87.9 420.3413.54 26.74 2.51 463.12 67160-10 napin cotton GMT T2 90.8 465.41 15.3221.78 2.69 505.21 67160-13 napin cotton GMT T2 92.1 3.98 0.00 502.7815.54 522.30  9979-94 control 0.8 0.8 4.27 0.00 510.20 17.15 531.62 9979-93 control 0.8 4.42 0.00 549.18 18.50 572.10  9979-91 control 0.84.43 0.00 480.59 14.35 499.38  9979-95 control 0.9 5.22 0.00 538.4819.08 562.78  9979-92 control 0.9 306.93 7.18 193.74 7.25 515.10 67170-3Brassica S8 GMT T2 59.6 77.8 364.13 8.20 151.34 5.92 529.59 67170-6Brassica S8 GMT T2 68.8 355.93 6.18 137.59 5.36 505.06 67170-2 BrassicaS8 GMT T2 70.5 381.42 8.51 142.79 6.09 538.82 67170-14 Brassica S8 GMTT2 70.8 372.06 5.24 130.94 4.04 512.28 67170-9 Brassica S8 GMT T2 72.6368.24 7.38 108.85 4.32 488.79 67170-1 Brassica S8 GMT T2 75.3 374.715.53 97.22 3.29 480.75 67170-15 Brassica S8 GMT T2 77.9 419.64 11.3988.39 4.20 523.61 67170-5 Brassica S8 GMT T2 80.1 408.32 3.44 88.98 6.94507.68 67170-11 Brassica S8 GMT T2 80.4 438.52 10.27 55.07 3.73 507.5967170-8 Brassica S8 GMT T2 86.4 452.28 12.04 49.76 2.65 516.72 67170-7Brassica S8 GMT T2 87.5 461.35 10.82 51.41 2.62 526.20 67170-4 BrassicaS8 GMT T2 87.7 458.39 10.45 17.75 1.16 487.76 67170-12 Brassica S8 GMTT2 94.0 5.31 0.00 528.79 20.48 554.59 1  9979 1.0 1.1 5.91 0.00 543.9621.53 571.40 2  9979 1.0 5.26 0.00 515.35 18.45 539.07 3  9979 1.0 6.520.00 509.65 19.20 535.37 4  9979 1.2 7.70 0.00 537.19 22.97 567.87 5 9979 1.4 5.21 0.00 511.12 19.85 536.17 6  9979 1.0 301.07 4.48 125.807.99 439.34   2-8 67159 = brassica P4 GMT T3 68.5 68.1 306.33 3.22169.37 8.75 487.68   2-3 67159 = brassica P4 GMT T3 62.8 320.26 6.05167.87 8.65 502.84   2-4 67159 = brassica P4 GMT T3 63.7 329.45 7.12169.63 9.21 515.41   2-2 67159 = brassica P4 GMT T3 63.9 329.53 5.80152.26 8.99 496.59   2-5 67159 = brassica P4 GMT T3 66.4 334.46 5.82145.10 8.16 493.54   2-6 67159 = brassica P4 GMT T3 67.8 335.46 4.25141.18 8.39 489.28   2-7 67159 = brassica P4 GMT T3 68.6 344.53 8.17145.61 9.24 507.54   2-1 67159 = brassica P4 GMT T3 67.9 401.15 5.4168.31 8.01 482.88   2-9 67159 = brassica P4 GMT T3 83.1 345.21 3.07161.54 11.71 521.53   4-2 67159 = brassica P4 GMT T3 66.2 89.2 431.506.46 56.16 6.72 500.83   4-9 67159 = brassica P4 GMT T3 86.2 445.25 5.6920.55 7.24 478.73   4-8 67159 = brassica P4 GMT T3 93.0 445.71 5.4820.58 6.60 478.36   4-3 67159 = brassica P4 GMT T3 93.2 446.77 7.7414.86 5.03 474.41   4-7 67159 = brassica P4 GMT T3 94.2 452.65 8.9649.76 7.52 518.89   4-4 67159 = brassica P4 GMT T3 87.2 454.02 8.0914.05 5.10 481.26   4-6 67159 = brassica P4 GMT T3 94.3 467.24 9.6511.93 4.93 493.75   4-1 67159 = brassica P4 GMT T3 94.6 517.68 12.9513.39 5.10 549.12   4-5 67159 = brassica P4 GMT T3 94.3 347.03 2.66155.38 8.28 513.35   7-5 67159 = brassica P4 GMT T3 67.6 81.9 350.320.48 132.12 8.20 491.12   7-7 67159 = brassica P4 GMT T3 71.3 352.481.50 141.14 8.26 503.37   7-2 67159 = brassica P4 GMT T3 70.0 367.651.04 134.34 7.75 510.78   7-8 67159 = brassica P4 GMT T3 72.0 372.230.00 125.08 7.40 504.71   7-6 67159 = brassica P4 GMT T3 73.8 454.167.27 10.99 3.38 475.80   7-4 67159 = brassica P4 GMT T3 95.5 464.63 6.0810.50 3.10 484.31   7-9 67159 = brassica P4 GMT T3 95.9 467.40 6.9911.11 3.82 489.32   7-1 67159 = brassica P4 GMT T3 95.5 474.28 8.2311.61 4.65 498.77   7-3 67159 = brassica P4 GMT T3 95.1 324.79 0.00179.06 11.83 515.68   11-7 67159 = brassica P4 GMT T3 63.0 82.2 334.920.00 175.60 11.84 522.35   11-2 67159 = brassica P4 GMT T3 64.1 352.840.00 170.23 12.16 535.22   11-5 67159 = brassica P4 GMT T3 65.9 425.544.66 49.26 5.84 485.30   11-3 67159 = brassica P4 GMT T3 87.7 427.095.61 61.10 6.38 500.18   11-4 67159 = brassica P4 GMT T3 85.4 448.326.34 12.02 4.67 471.35   11-6 67159 = brassica P4 GMT T3 95.1 462.497.21 42.46 7.43 519.59   11-1 67159 = brassica P4 GMT T3 89.0 464.304.97 12.86 5.43 487.55   11-9 67159 = brassica P4 GMT T3 95.2 469.004.57 16.21 5.08 494.86   11-8 67159 = brassica P4 GMT T3 94.8 427.197.33 43.05 4.39 481.96   4-9 67156 = napin GMT arab T3 88.6 94.0 429.833.85 47.80 3.09 484.57   4-8 67156 = napin GMT arab T3 88.7 442.62 8.9745.02 3.71 500.32   4-4 67156 = napin GMT arab T3 88.5 449.25 4.88 13.312.54 469.99   4-2 67156 = napin GMT arab T3 95.6 454.35 6.96 2.91 2.58466.79   4-5 67156 = napin GMT arab T3 97.3 459.55 7.20 2.75 1.43 470.94  4-6 67156 = napin GMT arab T3 97.6 467.64 9.17 5.77 2.51 485.09   4-367156 = napin GMT arab T3 96.4 469.22 7.89 9.04 3.43 489.58   4-1 67156= napin GMT arab T3 95.8 476.93 6.07 3.18 2.68 488.85   4-7 67156 =napin GMT arab T3 97.6 341.52 0.00 152.78 6.96 501.27   7-1 67156 =napin GMT arab T3 68.1 90.7 426.76 3.74 55.93 7.18 493.62   7-2 67156 =napin GMT arab T3 86.5 427.82 2.42 36.53 3.79 470.56   7-7 67156 = napinGMT arab T3 90.9 448.96 3.62 8.76 3.29 464.62   7-9 67156 = napin GMTarab T3 96.6 455.79 5.26 12.41 3.45 476.91   7-6 67156 = napin GMT arabT3 95.6 457.18 6.56 21.53 2.89 488.16   7-5 67156 = napin GMT arab T393.7 461.11 6.33 8.82 3.36 479.62   7-8 67156 = napin GMT arab T3 96.1462.08 7.10 16.36 3.59 489.14   7-4 67156 = napin GMT arab T3 94.5466.01 7.72 15.40 4.54 493.68   7-3 67156 = napin GMT arab T3 94.4 5.090.00 535.79 19.35 560.22  9979-81: @.0005. Control 0.9 5.37 0.00 534.9321.47 561.77  9979-81: @.0006. Control 1.0 327.76 22.52 156.62 9.37516.27 67158-2: @.0002. napin Cuphea GMT T3 63.5 85.2 384.99 24.97 92.367.82 510.14 67158-2: @.0001. napin Cuphea GMT T3 75.5 406.19 27.74 3.422.12 439.47 67158-2: @.0006. napin Cuphea GMT T3 92.4 424.62 22.33 34.406.92 488.27 67158-2: @.0009. napin Cuphea GMT T3 87.0 432.70 25.03 52.968.60 519.29 67158-2: @.0004. napin Cuphea GMT T3 83.3 443.67 25.50 46.418.22 523.80 67158-2: @.0003. napin Cuphea GMT T3 84.7 449.38 26.25 4.062.34 482.03 67158-2: @.0005. napin Cuphea GMT T3 93.2 449.63 25.26 2.171.84 478.89 67158-2: @.0008. napin Cuphea GMT T3 93.9 451.00 25.32 6.562.74 485.63 67158-2: @.0007. napin Cuphea GMT T3 92.9 312.62 22.03153.68 6.73 495.05 67158-4: @.0007. napin Cuphea GMT T3 63.1 75.7 326.5023.50 131.44 6.54 487.99 67158-4: @.0001. napin Cuphea GMT T3 66.9327.91 22.51 143.83 7.42 501.67 67158-4: @.0005. napin Cuphea GMT T365.4 331.65 24.40 137.74 7.20 500.98 67158-4: @.0009. napin Cuphea GMTT3 66.2 345.95 24.75 134.17 6.75 511.62 67158-4: @.0006. napin CupheaGMT T3 67.6 355.47 24.91 120.77 6.50 507.65 67158-4: @.0003. napinCuphea GMT T3 70.0 448.67 24.98 0.92 1.97 476.54 67158-4: @.0004. napinCuphea GMT T3 94.2 453.62 25.23 0.98 1.59 481.42 67158-4: @.0008. napinCuphea GMT T3 94.2 456.45 27.19 1.34 1.92 486.91 67158-4: @.0002. napinCuphea GMT T3 93.7 6.39 0.00 498.67 24.65 529.71  9979-81: @.0007.Control 1.2 6.65 0.00 520.22 19.20 546.08  9979-81: @.0008. Control 1.2325.71 19.95 154.88 8.09 508.64 67158-9: @.0007. napin Cuphea GMT T364.0 68.4 330.27 21.90 154.36 8.08 514.61 67158-9: @.0005. napin CupheaGMT T3 64.2 347.97 22.33 129.57 6.54 506.41 67158-9: @.0004. napinCuphea GMT T3 68.7 351.68 22.59 122.64 6.96 503.87 67158-9: @.0006.napin Cuphea GMT T3 69.8 353.74 22.51 118.23 6.90 501.38 67158-9:@.0001. napin Cuphea GMT T3 70.6 354.17 23.30 137.47 7.50 522.4467158-9: @.0002. napin Cuphea GMT T3 67.8 358.21 21.84 132.99 6.76519.80 67158-9: @.0009. napin Cuphea GMT T3 68.9 362.74 22.40 114.966.69 506.79 67158-9: @.0008. napin Cuphea GMT T3 71.6 362.98 24.28124.73 6.50 518.49 67158-9: @.0003. napin Cuphea GMT T3 70.0 403.3526.19 33.39 3.08 466.02 67158-14: @.0003. napin Cuphea GMT T3 86.6 90.0416.91 26.96 34.74 3.21 481.83 67158-14: @.0002. napin Cuphea GMT T386.5 423.10 22.19 36.04 3.17 484.50 67158-14: @.0008. napin Cuphea GMTT3 87.3 424.87 26.52 4.48 1.62 457.49 67158-14: @.0004. napin Cuphea GMTT3 92.9 428.75 23.34 24.92 5.13 482.14 67158-14: @.0009. napin CupheaGMT T3 88.9 433.96 30.08 5.32 2.24 471.61 67158-14: @.0001. napin CupheaGMT T3 92.0 434.51 29.70 20.34 1.90 486.44 67158-14: @.0005. napinCuphea GMT T3 89.3 435.86 23.44 3.27 1.75 464.33 67158-14: @.0006. napinCuphea GMT T3 93.9 440.46 23.40 10.67 2.27 476.80 67158-14: @.0007.napin Cuphea GMT T3 92.4

EXAMPLE 3

[0382] Computer programs are used to predict the chloroplast targetingpeptide cleavage sites of the plant GMT proteins. The predictions ofCTPs by using two programs: “Predotar” and “ChloroP” (Center forBiological Sequence Analysis, Lyngby, Denmark) are as follows

[0383] 1) Program: Predotar Sequence ID Score Cut Site P-Value Gossypium4.56 49 * 50 3.0496E+07 Brassica 2.27 51 * 52 2.3192E+05 Cuphea 1.96undetermined 2.7934E−01

[0384] 2) Chloroplast Target Peptide Prediction Results

[0385] Number of Query Sequences: 5 Name Length Score cTP CS-scorecTP-length Arabidopsis 348 0.587 Y 7.834 50 Gossypium 345 0.580 Y 4.11648 Brassica 347 0.581 Y 8.142 51 Cuphea 376 0.573 Y 1.746 64 Zea mays352 0.560 Y 4.808 48

[0386] Based on this information GMT proteins from plant sources areengineered to remove the predicted chloroplast target peptides to allowfor the expression of the mature protein in E. coli. In order for theseproteins to be expressed in a prokaryotic expression system, an aminoterminal methionine is required. This can be accomplished, for example,by the addition of a 5′ ATG. A methionine is added to each of thefollowing amino acid sequences, which are expressed in E. coli withdetectable GMT activity (SEQ ID NOs: 33-38 each have the addedmethionine as the first amino acid in the sequence): Mature S8 Brassicanapus GMT protein as expressed in E. coli (SEQ ID NO: 33); Mature P4Brassica napus GMT protein as expressed in E. coli (SEQ ID NO: 34);Mature Cuphea pulcherrima GMT protein as expressed in E. coli (SEQ IDNO: 35); Mature Gossypium hirsutum GMT protein as expressed in E. coli(SEQ ID NO: 36); Mature Tagetes erecta (Marigold) GMT protein asexpressed in E. coli (SEQ ID NO:37); Mature Zea mays (Corn) GMT proteinas expressed in E. coli (SEQ ID NO: 38).

[0387] Constructs are prepared to direct expression of the mature P4 andS8 Brassica napus, Cuphea pulcherrima, Gossypium hirsutum, Tageteserecta, and Zea mays GMT sequences in a prokaryotic expression vector.The mature protein-coding region of each GMT with the aminotermianlmethionine, as described previously, is amplified from plasmid DNA usingthe following species specific oligonucleotide primers in the polymerasechain reaction (PCR). Components of each 100 μl PCR reaction at finalconcentration consisted of: 1.0 μl genomic DNA or 1.0 μl plasmid DNAdiluted 1:20 with water, as appropriate, 1×Buffer 2 (Expand™ HighFidelity PCR System, Roche Molecular Biochemicals), 200 μM dNTPs, 300 nMeach, synthetic oligonucleotide primers, and 0.8 μl Expand™ HighFidelity Polymerase (Roche Molecular Biochemicals, Indianapolis, Ind.).

[0388] “Touchdown” cycling conditions consisted of a pre-incubation for3 min at 94° C., during which the Expand polymerase is spiked into themix. The product is then amplified with 15 cycles of denaturation at 94°C. for 45 sec, annealing at 70° C. for 30 sec, and elongation at 72° C.for 1.5 min. The annealing temperature is decreased by 1° C. per cyclefor each of the previous 15 cycles. An additional 15 cycles followed,consisting of 94° C. for 45 sec, 55° C. for 30 sec, and 72° C. for 1.5min, followed by a 7 min at 72° C.

[0389] A mature S8 Brassica GMT coding sequence is amplified frompMON67170 using the synthetic oligonucleotide primers: #16765 (SEQ IDNO: 59) and #16654 (SEQ ID NO: 60).

[0390] A mature P4 Brassica GMT coding sequence is amplified frompMON67159 using the synthetic oligonucleotide primers: #16765 (SEQ IDNO: 59) and #16654 (SEQ ID NO: 60).

[0391] A mature Cuphea pulcherrima GMT coding sequence is amplified frompMON67158 using the synthetic oligonucleotide primers: #16763 (SEQ IDNO: 61) and #16659 (SEQ ID NO: 62).

[0392] A mature Gossypium hirsutum GMT coding sequence is amplified frompMON67160 using the synthetic oligonucleotide primers: #16764 (SEQ IDNO: 63) and #16682 (SEQ ID NO: 64).

[0393] A mature Tagetes erecta GMT coding sequence is amplified from theEST clone LIB3100-001-Q1-M1-E2 using the synthetic oligonucleotideprimers: #16766 (SEQ ID NO: 65) and #16768 (SEQ ID NO: 66).

[0394] A mature Zea mays GMT coding region is amplified from the ESTclone LIB3689-262-Q1-K1-D6 using the synthetic oligonucleotide primers:5′GGG GAC AAG TTT GTA CAA AAA AGC AGG CTT AGA AGG AGA TAG AAC CAT GGCCTC GTC GAC GGC TCA GGC CC3′ (SEQ ID NO: 73) and 5′GGG GAC CAC TTT GTACAA GAA AGC TGG GTC CTG CAG GCT ACG CGG CTC CAG GCT TGC GAC AG (SEQ IDNO: 74).

[0395] A GMT coding region from Nostoc punctiforme (ATCC 29133) isamplified from genomic DNA. Genomic DNA is isolated from 3 day culturesof the cyanobacteria according to the procedure of Chisholm (CYANONEWS,Vol. 6. No. 3 (1990)). Cultures are centrifuged and the supernatentdiscarded. Pellets are suspended in 400 μl TES (TES: 2.5 ml of 1 M Tris,pH 8.5; 5 ml of 5 M NaCl; 5 ml of 500 mM EDTA, bring volume to 500 ml.)To the suspended pellet, 100 μl lysozyme (50 mg/ml) is added and thesuspension incubated for 15 minutes at 37° C. with occasional mixing. Tothis, 50 μl sarkosyl (10%) is added. Protein is extracted by adding 600μl phenol and incubating at room temperature with gentle shaking. Thephases are separated by centrifugation and the aqueous phase istransferred to a new tube. RNase is added to a final concentration of1.0 mg/ml and the solution is incubated for 15 minutes at 37° C. To thissolution 100 μl NaCl (5M), 100 μl CTAB/NaCl (CTAB/NaCl: To 80 ml ofwater, add 4.1 g of NaCl, then 10 g CTAB, heat to 65° C. to dissolve,bring volume to 100 ml), and 600 μl chloroform are added and thesolution incubated 15 minutes at room temperature with gentle shaking.The phases are separated by centrifugation and the aqueous phase istransferred to a new tube. 700 μl isopropanol is added to precipitateDNA. The sample is centrifuged for 15 minutes at 14,000 rpms in amicro-centrifuge to pellet genomic DNA. The pellet is rinsed with 70%ethanol, dried briefly in a Speedvac and the genomic DNA is suspended in100 μl TE. DNA concentration, as determined by spectrophotometry, is 79μg/ml.

[0396] Nostoc GMT amplification reactions contained 79 ng genomic DNA,2.5 μl 20×dNTPs 2.5 μl of each of the following primers: 5′GGG GAC AAGTTT GTA CAA AAA AGC AGG CTT AGA AGG AGA TAG AAC CAT GAG TGC AAC ACT TTACCA GCA AAT TC 3′ (SEQ ID NO: 67) and 5′GGG GAC CAC TTT GTA CAA GAA AGCTGG GTC CTA CTA CTT ATT GCC GCA CAG TAA GC 3′ (SEQ ID NO: 68), 5 μl10×PCR buffer 2 or 3, and 0.75 μl Expand High Fidelity DNA Polymerase.PCR conditions for amplification are as follows: 1 cycle of 94° C. for 2minutes, 10 cycles of 94° C.—15 seconds; 55° C.—30 seconds; and 72°C.—1.5 minutes, 15 cycles of 94° C.—15 seconds; 55° C.—30 seconds; and72° C.—1.5 minutes adding 5 seconds to the 72° C. extension with eachcycle, 1 cycle of 72° C. for 7 minutes. After amplification, samples arepurified using a Qiagen PCR cleanup column, suspended in 30 μl water and10 μl are visualized on an agarose gel.

[0397] GMT and MT1 coding sequences are amplified from genomic DNA fromthe cyanobacterium Anabaena species (ATCC 27893). DNA used for PCRamplification of Anabaena GMT and MT1 is isolated by collecting pelletsfrom 3 day old cyanobacteria cultures by centrifugation. The pellet iswashed with 1 ml PBS to remove media. The suspension is centrifuged andthe supernatent is discarded. The pellet is resuspended in 1 ml of waterand boiled for 10 minutes. Anabaena amplification reactions contained 10μl boiled Anabaena extract, 2.5 μl 20×dNTPs 2.5 μl of each primer, 5 μl10×PCR buffer 2 or 3, and 0.75 μl Expand High Fidelity DNA Polymerase.PCR conditions for amplification are as follows: 1 cycle of 94° C. for 2minutes, 10 cycles of 94° C.—15 seconds; 55° C.—30 seconds; and 72°C.—1.5 minutes, 15 cycles of 94° C.—15 seconds; 55° C.—30 seconds; and72° C.—1.5 minutes adding 5 seconds to the 72° C. extension with eachcycle, 1 cycle of 72° C. for 7 minutes. After amplification, samples arepurified using a Qiagen PCR cleanup column, suspended in 30 μl water and10 μl are visualized on an agarose gel.

[0398] Anabaena species GMT coding sequence is amplified using thesynthetic oligonucleotide primers: 5′GGG GAC AAG TTT GTA CAA AAA AGC AGGCTT AGA AGG AGA TAG AAC CAT GAG TGC AAC ACT TTA CCA ACA AAT TCA G 3′(SEQ ID NO: 69) and 5′GGG GAC CAC TTT GTA CAA GAA AGC TGG GTC CTA TCACTT ATC CCC ACA AAG CAA CC 3′ (SEQ ID NO: 70).

[0399] Anabaena species MT1 coding sequence is amplified using thesynthetic oligonucleotide primers: 5′GGG GAC AAG TTT GTA CAA AAA AGC AGGCTT AGA AGG AGA TAG AAC CAT GAG TTG GTT GTT TTC TAC ACT GG 3′ (SEQ IDNO: 71) and 5′GGG GAC CAC TTT GTA CAA GAA AGC TGG GTC CTA TTA CTT TTGAGC AAC CTT GAT CG3′ (SEQ ID NO: 72).

[0400] The resulting PCR products are subcloned into pDONR™201 (LifeTechnologies, A Division of Invitrogen Corp., Rockville, Md.) using theGATEWAY cloning system (Life Technologies, A Division of InvitrogenCorp., Rockville, Md.) and labeled pMON67180 (mature S8 Brassica napusGMT), pMON68757 (mature P4 Brassica napus GMT), pMON68755 (mature Cupheapulcherrima GMT), pMON68756 (mature Gossypium hirsutum GMT), pMON68758(mature Tagetes erecta GMT), pMON67182 (mature Zea mays GMT), pMON67520(Nostoc punctiforme GMT), pMON67518 (Anabaena species GMT), andpMON67517 (Anabaena species MT1). Double stranded DNA sequence isobtained to verify that no errors are introduced by the PCRamplification.

[0401] For functional testing GMT and MT1 sequences are then recombinedbehind the T7 promoter in the prokaryotic expression vector pET-DEST42(FIG. 1) (Life Technologies, A Division of Invitrogen Corp., Rockville,Md.) using the GATEWAY cloning system (Life Technologies, A Division ofInvitrogen Corp., Rockville, Md.) according to the manufacturer'sprotocol. The resulting expression vectors are labeled pMON67181 (matureS8 Brassica napus GMT), pMON67172 (mature P4 Brassica napus GMT),pMON67173 (mature Cuphea pulcherrima GMT), pMON67171 (mature Gossypiumhirsutum GMT), pMON67177 (mature Tagetes erecta GMT), pMON67176 (Nostocpunctiforme GMT), pMON67175 (Anabaena species GMT), pMON67174 (Anabaenaspecies MT1), and pMON67183 (Zea Mays GMT) (see also table 6). TABLE 6Bacterial expression vectors for functional testing ofmethyltransferases Construct I.D. Gene Source of Gene ModificationspMON67171* GMT Gossypium hirsutum Mature protein pMON67172* GMT Brassicanapus P4 Mature protein pMON67173* GMT Cuphea pulcherrima Mature proteinpMON67174 MT1 Anabaena pMON67175 GMT Anabaena pMON67176 GMT NostocpMON67177* GMT Tagetes erecta Mature protein pMON67181* GMT Brassicanapus S8 Mature protein pMON67183* GMT Zea mays Mature protein

EXAMPLE 4

[0402] Bacterial expression plasmids listed in Table 6 are transformedinto expression host cells (BL21 (DE3)(Stratagene, La Jolla, Calif.))prior to growth and induction. A 100 mL LB-culture with the appropriateselection antibiotic (mg/mL carbenicillin) is inoculated with anovernight starter culture of cell transformants to an OD₆₀₀ of 0.1 andgrown at 25° C., 250 rpm to an OD₆₀₀ of 0.6. The cells are then inducedby adding IPTG to a final concentration of 0.4 mM and incubating forthree hours at 25° C. and 200 rpm. Cultures are transferred to 250 mLpolypropylene centrifuge tubes, chilled on ice for five minutes, andharvested by centrifugation at 5000×g for ten minutes. The cell pelletis stored at −80° C. after thoroughly aspirating off the supernatant.

[0403] Methyltransferase activity is measured in vitro using amodification of the method described by d'Harlingue et al., 1985d'Harlingue and Camara, J. Biol. Chem. 260(28):15200-3 (1985). The cellpellet is thawed on ice and resuspended in 4 mL of extraction buffer (10mM HEPES-KOH pH 7.8, 5 mM DTT (dithiothriotol), 1 mM AEBSF(4-(2-aminoethyl) benzenesulfonyl fluoride), 0.1 μM aprotinin, 1 μg/mLleupeptin). Cells are disrupted using a French press. Each cellsuspension is run through the pressure cell twice at 20,000 psi. Tritonx-100 is added to a final concentration of 1% and the cell homogenate isincubated on ice for one hour before centrifugation at 5000 g for tenminutes at 4° C. The supernatant is transferred to fresh eppendorf tubesfor methyltransferase activity analysis.

[0404] Enzyme assays are performed in assay buffer containing 50 mMTris-HCl pH 7.0 (pH 8.0 for MT1), 5 mM DTT, 100 μM substrate(γ-tocopherol or γ-tocotrienol for GMT (Calbiochem-NovabiochemCorporation, San Diego, Calif.); 2-methylphytylplastoquinol (racemicmixture) (2-methylphytylplastoquinol and2,3-dimethyl-5-phytylplastoquinol are synthesized as described by Solland Schultz 1980 (Soll, J., Schultz, G., 1980,2-methyl-6-phytylplastoquinol and 2,3-dimethyl-5-phytyl-plastoquinol asprecursors of tocopherol synthesis in spinach chloroplasts,Phytochemistry 19:215-218) for MT1 and TMT2), 0.1 μCi ¹⁴C-SAM (48μCi/μmole, ICN Biomedicals, Aurora, Ohio), and 0.5% tween 80 (forsubstrate solubility) in a final volume of 1 mL. Reactions are preparedin 10 mL polypropylene culture tubes by first adding the substrate fromconcentrated stocks dissolved in hexane and evaporating off the hexaneunder nitrogen gas flow. Tween 80 is added directly to the substratebefore adding the remainder of the assay buffer less the SAM. Crude cellextract is added to the assay mix in 50 μL volumes and the timedreactions are initiated by adding SAM. Reactions are vortexed thoroughlyto dissolve all of the detergent into the mix and then incubated at 30°C. in the dark for 30 minutes.

[0405] The reactions are transferred to 15 mL screw-capped glass tubeswith teflon-coated caps prior to quenching and phase extracting with 4mL of 2:1 chloroform/methanol containing 1 mg/mL of butylatedhydroxytoluene (BHT for stability of the end product). These are thenvortexed for at least 30 seconds and centrifuged at 800×g for 5 minutesto separate the layers. If necessary, 1 mL of 0.9% NaCl is added toimprove the phase separation (emulsions may form because the enzyme isadded as a crude extract). The organic phase (bottom layer) of eachphase extraction is transferred to a fresh 15 mL glass tube andevaporated completely under nitrogen gas flow. The reaction products arethen dissolved in 200 μL of ethanol containing 1% pyrogalol and vortexedfor at least 30 seconds. This is filtered through a 0.2 μm filter(Whatman PTFE) into glass inserts contained within light protected LCvials for HPLC analysis.

[0406] The HPLC (HP 1100) separation is carried out using a normal phasecolumn (Agilent Zorbax Sil, 5 μm, 4.6×250 mm) with 1.5 mL/minuteisocratic flow of 10% methyl-t-butyl-ether in hexane over a period of 14minutes. Samples are injected onto the column in 50 μl volumes.Quantitation of ¹⁴C-labeled reaction products is performed using a flowscintillation counter (Packard 500TR). Methyltransferase activities arecalculated based on a standard curve of D-α-[5-methyl-¹⁴C]-tocopherol(Amersham-Pharmacia, 57 mCi/mmol).

[0407] The assay results confirm γ-tocopherol methyltransferase activityfor all GMT gene candidates listed in table 6, except for the BrassicaP4 gene (FIG. 17).

[0408] The MT1 assay results (FIG. 33) indicated2-methylphytylplastoquinol methyltransferase activity with the AnabaenaMT1 expression product. FIGS. 18, 19, and 20 represent HPLCchromatograms of the MT1 assay carried out with recombinant expressedAnabaena MT1, with recombinant Anabaena MT1 without2-methylphytyl-plastoquinol substrate, and an assay performed with peachloroplast extract as a positive control for the MT1 assay,respectively.

[0409] The Anabaena, corn, and cotton GMTs are chosen for the purpose ofcomparing enzymes from microbial and monocotyledon sources versusdicotyledon plant sources for methyltransferase activity withγ-tocotrienol. Assays are run in duplicate with γ-tocopherol assays runin parallel as controls. In both cases 100 μM of substrate is used, withthe substrate as the only difference in assay conditions. The monocotGMT showed comparable methyltransferase activity with γ-tocopherol andγ-tocotrienol. In contrast the bacterial and the dicot GMT aresubstantially less active with γ-tocotrienol. The results of thisexperiment are summarized in FIG. 34.

EXAMPLE 5

[0410] Seed specific expression of GMT in Brassica is obtained bylinking the Arabidopsis thaliana, ecotype Columbia gene to the napinpromoter as described here. Poly A+ RNA is isolated from Arabidopsisthaliana, ecotype Columbia using an adapted biotin/streptavadinprocedure based on a mRNA Capture Kit” (Roche Molecular Biochemicals,Indianapolis, Ind.). Young leaf tissue is homogenized in CTAB buffer(5OmM Tris-HCl pH9, 0.8M NaCl, 0.5% CTAB, 10 mM EDTA), extracted withchloroform and pelleted. As set forth in the manufacturer'sinstructions, the soluble phase is hybridized to biotin-labeledoligo-dT, immobilized on streptavadin-coated PCR tubes and washed. Firststrand cDNA is synthesized using the “1^(st) strand cDNA synthesis kitfor RT-PCR” (Roche Molecular Biochemicals, Indianapolis, Ind.). cDNAsynthesis is performed according to the manufacturer's protocol andfollowed by RNase digestion (0.5 units RNase in 48 μl for 30 min.).

[0411]Arabidopsis thaliana, ecotype Columbia is amplified using primers#16562 Arab GMT Forward-Not 5′ GCG GCC GCA CAA TGA AAG CAA CTC TAG CAGCAC CCT C 3′ (SEQ ID NO: 77) and #16563 Arab GMT Reverse-Sse 5′ CCT GCAGGT TAG AGT GGC TTC TGG CAA GTG ATG 3′ (SEQ ID NO: 78) and the “ExpandHigh Fidelity PCR System (Roche Molecular Biochemicals, Indianapolis,Ind.). A GMT gene is PCR-amplified for 30 cycles using a “touchdown”cycling profile: 3 min incubation at 94° C., followed by 15 cycles of 45seconds denaturation at 94° C., 30 seconds annealing at 60° C. and 2 minextensions at 72° C. Primers are designed to add a NotI/Kozak site and a3′ Sse83871 site.

[0412] The PCR product is desalted using a Pharmacia Microspin S-400 HRColumn (Pharmacia, Uppsala, Sweden). The purified fragment is insertedinto pCR2.1 using a TOPO TA Cloning Kit (Invitrogen, Carlsbad, Calif.)resulting in the formation of pMON67155. The nucleotide sequence of theinsert, Arabidopsis thaliana, ecotype Columbia GMT is confirmed by DNAsequencing. The GMT insert is excised from pMON67155 by NotI/Sse8371digestion. Restriction enzymes are removed using StrataClean Resin(Stratagene, La Jolla, Calif.) and passed through a Microspin S-400 HRColumn (Pharmacia, Uppsala, Sweden). The fragment is ligated intoNotI/Sse83871 digested, identically treated pMON11307, resulting in theformation of the binary vector pMON67157 (FIG. 13).

[0413] The plant binary construct described above is used in Brassicanapus plant transformation to direct the expression of thegamma-methyltransferases in the embryo. The vector is transformed intoABI strain Agrobacterium cells by the method of Holsters et al., Mol.Gen. Genet. 163:181-187(1978). Brassica plants maybe obtained byAgro-bacterium-mediated transformation as described by Radke et al.Plant Cell Reports 11: 499-505 (1992) and WO 00/61771. The tocopherollevel and composition of the seed from transgenic plants is analyzedusing the method set forth in example 6.

[0414] Results of Brassica transformation are shown in FIG. 24, which isa graph representing the seed α-tocopherol levels for varioustransformants. Table 7 represents transformation data from variouslines. TABLE 7 ng α ng β ng γ ng δ ng total toco./mg toco./mg toco./mgtoco./mg toco./mg % Avg. % seed seed seed seed seed Line Number AlphaAlpha Description 165.07 0.00 139.34 5.33 309.74 Control - Empty VectorR1 53.3 44.1 Control 102.41 0.00 189.34 3.76 295.50 Control R1 34.7Control 126.90 0.00 229.27 6.64 362.81 Control R1 35.0 Control 139.090.00 230.64 5.97 375.70 Control R1 37.0 Control 137.88 0.00 173.73 4.36315.97 Control R1 43.6 Control 203.16 0.00 126.41 2.74 332.31 Control R161.1 Control 113.75 0.00 187.68 5.86 307.29 Arabidopsis GMT in Canola R137.0 87.1 PMON67157-10 197.02 0.00 137.48 4.50 338.99 Arabidopsis GMT inCanola R1 58.1 PMON67157-9 201.11 0.00 134.65 6.52 342.28 ArabidopsisGMT in Canola R1 58.8 PMON67157-5 212.78 0.00 92.97 3.36 309.11Arabidopsis GMT in Canola R1 68.8 PMON67157-4 240.49 0.00 53.44 1.77295.70 Arabidopsis GMT in Canola R1 81.3 PMON67157-6 231.63 0.00 49.460.00 281.09 Arabidopsis GMT in Canola R1 82.4 PMON67157-25 234.90 0.0045.91 1.03 281.84 Arabidopsis GMT in Canola R1 83.3 PMON67157-20 334.070.00 57.69 1.65 393.41 Arabidopsis GMT in Canola R1 84.9 PMON67157-27345.00 0.00 36.75 2.23 383.99 Arabidopsis GMT in Canola R1 89.8PMON67157-21 286.02 0.00 1.04 1.61 288.67 Arabidopsis GMT in Canola R199.1 PMON67157-2 387.23 0.00 0.16 1.64 389.03 Arabidopsis GMT in CanolaR1 99.5 PMON67157-3 322.59 0.00 0.68 0.66 323.93 Arabidopsis GMT inCanola R1 99.6 PMON67157-8 331.27 0.00 0.46 0.61 332.34 Arabidopsis GMTin Canola R1 99.7 PMON67157-1 322.34 0.00 0.00 0.62 322.97 ArabidopsisGMT in Canola R1 99.8 PMON67157-24 316.73 0.00 0.51 0.00 317.24Arabidopsis GMT in Canola R1 99.8 PMON67157-13 357.05 0.00 0.24 0.00357.29 Arabidopsis GMT in Canola R1 99.9 PMON67157-17 310.97 0.00 0.170.00 311.13 Arabidopsis GMT in Canola R1 99.9 PMON67157-22 324.07 0.000.00 0.00 324.07 Arabidopsis GMT in Canola R1 100.0 PMON67157-23 367.840.00 0.00 0.00 367.84 Arabidopsis GMT in Canola R1 100.0 PMON67157-28438.54 0.00 0.00 0.00 438.54 Arabidopsis GMT in Canola R1 100.0PMON67157-30

EXAMPLE 6

[0415] Seed specific expression of GMT in soy is obtained by linking theArabidopsis thaliana, ecotype Columbia GMT gene with different types ofseed specific promoters as described here. Total RNA is isolated fromArabidopsis leaf tissue (ecotype Columbia) using the Qiagen “RNeasyplant mini kit” (Qiagen Inc., Valencia, Calif.). First strand cDNAsynthesized using the “1^(st) strand cDNA synthesis kit for RT-PCR” fromBoehringer Mannheim. RNA isolation and cDNA synthesis is performedaccording to the manufacturer protocols.

[0416] The Arabidopsis GMT is amplified using primers “GMT-ara 5′ CATGCC ATG GGA ATG AAA GCA ACT CTA GCA G” (SEQ ID NO: 75) and “GMT-ara 3′GTC AGA ATT CTT ATT AGA GTG GCT TCT GGC AAG” (SEQ ID NO: 76) and theBoehringer Mannheim “Expand™ High Fidelity PCR System”. The GMT gene isPCR-amplified by 30 cycles under the following conditions: 5 minincubation at 95° C., followed by 30 cycles of 1 min at 95° C., 1 minannealing at 58° C. and 2 min extension at 72° C. These reactions arefollowed by 5 min incubation at 72° C. The primers are designed to add amethionine and a glycin to the N-terminus of the GMT protein.

[0417] The PCR products are EcoRI and NcoI digested and gel purifiedusing the Qiagen “Qiaquick Gel Extraction Kit” (Qiagen Inc., Valencia,Calif.). Purified fragments are ligated into EcoRI/NocI digested and gelpurified pET30 (Novagen, Madison, Wis.) and pSE280 (Invitrogen,Carlsbad, Calif.) resulting in the formation of pMON26592 (FIG. 3) andpMON26593 (FIG. 4), respectively. Subsequently the Arabidopsis GMTsequence is confirmed. During the sequencing procedure it is found thatthe cloned sequence from the Columbia ecotype exhibited two nucleotidechanges compared to the Arabidopsis thaliana GMT sequence published inWO 99/04622 (position 345, change from C to T; position 523,substitution from T to G). While the first substitution is a silentmutation, the second nucleotide change resulted in an amino acid changefrom serine to alanine.

[0418] For generation of a GMT plant transformation vector under p7Spromoter control, a GMT is excised as a BglII/EcoRI fragment frompMON26592, gel purified, and cloned into a BglII/EcoRI digested and gelpurified vector containing a p7S expression cassette resulting in theformation of the shuttle vector pMON36500 (FIG. 6). The p7S::GMT_(At)expression cassette is excised from pMON36500 by PstI digest, the endsare filled in by T4 DNA polymerase treatment, gel purified, and clonedinto SmaI digested, alkaline phosphatase treated and gel purifiedpMON38207R, resulting in the formation of the binary vector pMON36503.

[0419] An NcoI/EcoRI digested, gel purified GMT excised from pMON26592is ligated into an NcoI/EcoRI digested vector harboring a pARC5-1expression cassette, resulting in the formation of the shuttle vectorpMON36502. The pARC5-1::GMTAt expression cassette is excised frompMON35502 by NotI digest, blunt ends are generated by treatment withKlenow fragment, the fragment is gel purified, and ligated into Sma Idigested, alkaline phosphatase treated and gel purified pMON38207R. Theresulting binary vector is designated pMON36505.

[0420] An arcelin 5 promoter harbors 6 ATG start codons at the 5′sequence located in different reading frames (Goosens et al., PlantPhysiol. (1999), 120(4), 1095-1104, Goosens et al., FEBS Lett. (1999),456(1), 160-164.). To decrease the risk of interference of these startcodons during gene expression, 4 of these putative translational startsites are deleted. Deletion of 4 ATG codons is achieved by PCR, usingprimers Parc5′ (5′-CCA CGT GAG CTC CTT CCT CTT CCC-3′) (SEQ ID NO: 79)and Parc3′ (5′-GTG CCA TGG CAG ATC TGA TGA TGG ATT GAT GGA-3′) (SEQ IDNO: 80). Primer Parc3′ is designed to hybridize to the Arcelin 5promoter sequence at the translational start site and delete 4 of the 6ATG codons. PCR is performed using pMON55524 (FIG. 5) as template DNAand the Boehringer Mannheim PCR Core Kit in 30 PCR cycles under thefollowing conditions: 5 min incubation at 95° C., followed by 30 cyclesof 1 min at 95° C., 1 min annealing at 60° C. and 40 second extension at72° C. These reactions are followed by 5 min incubation at 72° C. Theresulting approximately 360 bp PCR product is digested with SalI andNcoI, gel purified and cloned into SalI/NcoI digested and gel purifiedpMON55524, resulting in the formation of pMON36501 (FIG. 7). A DNAsequence of the cloned PCR product is confirmed by DNA sequencing. A GMTexpression cassette using the modified promoter is assembled by ligatingthe backbone of SmaI/NcoI digested and gel purified pMON36501 with aGMTAt::Arcelin 5 3′ terminator fusion obtained from SmaI/NcoI digestedgel purified pMON36502 (FIG. 8). The resulting shuttle vector isdesignated pMON36504 (FIG. 10). A binary vector (pMON36506) harboring aGMT expression cassette under the control of the modified arcelin 5promoter is generated by cloning the NotI digested, Klenow fragmenttreated (for blunt end generation), gel purified GMT expression cassetteinto gel purified SmaI digested alkaline phosphatase treated, and gelpurified pMON38207R vector backbone (5′-GAG TGA TGG TTA ATG CAT GAA TGCATG ATC AGA TCT GCC ATG GTC CGT CCT-3′ (SEQ ID NO: 81)(original DNAsequence at the translational start site of the Arcelin 5promoter—pARC5-1)(5′-GAG TGA TGG TTA ATC CAT CAA TCC ATC ATC AGA TCT GCCATG GTC CGT CCT-3′)(SEQ ID NO: 82) (DNA sequence at the translationalstart site of the mutated Arcelin 5 promoter—pARC5-1M))

[0421] GMT expression vectors pMON36503 (FIG. 9), pMON36505 (FIG. 11)and pMON36506 (FIG. 12) are transformed into the soybean line A3244using Agrobacterium mediated transformation. See, for example themethods described by Fraley et al., Bio/Technology 3:629-635 (1985) andRogers et al., Methods Enzymol. 153: 253-277 (1987). Ten bulked seedsfrom the R₁ generation are ground and the resulting soy meal is used fortocopherol analysis. Twenty five to forty mg of the soy meal is weighedinto a 2 mL micro tube, and 500 μl 1% pyrogallol (Sigma Chemicals, St.Louis, Mo.) in ethanol containing 5 μg/mL tocol, is added to the tube.The sample is shaken twice for 45 seconds in a FastPrep (Bio101/Savant)using speed 6.5. The extract is then filtered (Gelman PTFE acrodisc 0.2μm, 13 mm syringe filters, Pall Gelman Laboratory Inc, Ann Arbor, Mich.)into an autosampler tube. HPLC is performed on a Zorbax silica HPLCcolumn, 4.6 mm×250 mm (5 μm) with a fluorescent detection using aHewlett Packard HPLC (Agilent Technologies). Sample excitation isperformed at 290 nm, and emission is monitored at 336 nm. Tocopherolsare separated with a hexane methyl-t-butyl ether gradient using aninjection volume of 20 μl, a flow rate of 1.5 ml/min, and a run time of12 min (40° C.). Tocopherol concentration and composition is calculatedbased on standard curves for α, β, γ and δ-tocopherol using Chemstationsoftware (Agilent Technologies, Palo Alto, Calif.) As shown in FIGS.14-16, several lines from each construct completely or substantiallyconverted δand γ-tocopherol, leaving a and β-tocopherol as the onlydetectable tocopherol isomers.

EXAMPLE 7

[0422] Canola, Brassica napus, or soybean plants are transformed with avariety of DNA constructs using Agrobacterium mediated transformation.Two sets of DNA constructs are produced. The first set of constructs are“single gene constructs”. Each of the following genes is inserted into aseparate plant DNA construct under the control of a seed specificpromoter such as the arcelin 5, 7S α or napin promoter (Kridl et al.,Seed Sci. Res. 1:209:219 (1991) (Keegstra, Cell 56(2):247-53 (1989);Nawrath, et al., Proc. Natl. Acad. Sci. U.S.A. 91:12760-12764 (1994)): abifunctional prephenate dehydrogenase such as the E. herbicola or the E.coli tyrA gene (Xia et al., J. Gen. Microbiol. 138:1309-1316 (1992)), aphytylprenyltransferase such as the slr/736 (in Cyanobase(www.kazusa.or.jp/cyanobase)) or the ATPT2 gene (Smith et al., Plant J.11: 83-92 (1997)), a 1 -deoxyxylulose 5-phosphate synthase such as theE. coli dxs gene (Lois et al., Proc. Natl. Acad. Sci. U.S.A. 95(5):2105-2110 (1998)), a 1-deoxyxylulose 5-phosphate reductoisomerase(dxr) gene (Takahashi et al. Proc. Natl. Acad. Sci. U.S.A. 95 (17),9879-9884 (1998)), a p-hydroxyphenylpyruvate dioxygenase, such as theArabidopsis thaliana HPPD gene (Norris et al., Plant Physiol.117:1317-1323 (1998)), a geranylgeranylpyrophosphate synthase gene suchas the Arabidopsis thaliana GGPPS gene (Bartley and Scolnik, PlantPhysiol. 104:1469-1470 (1994)), a transporter such as the AANT1 gene(Saint Guily, et al., Plant Physiol., 100(2):1069-1071 (1992)), a GMTgene, an MT1 gene, and a tocopherol cyclase such as the slr1737 gene (inCyanobase (www.kazusa.or.jp/cyanobase) or its Arabidopsis ortholog(PIR_T04448)), a isopentenylpyrophosphate isomerase gene (IDI), and anantisense construct for homogentisic acid dioxygenase (Sato et al., J.DNA Res. 7 (1):31-63 (2000))). The products of the genes are targeted tothe plastid by natural plastid target peptides present in the transgene, or by an encoded plastid target peptide such as CTP1. Eachconstruct is transformed into at least one canola, Brassica napus andsoybean plant. Plants expressing each of these genes are selected toparticipate in additional crosses. Crosses are carried out for eachspecies to generate transgenic plants having one or more of thefollowing combination of introduced genes: tyrA, slr1736, ATPT2, dxs,dxr, GGPPS, HPPD, GMT, MT1, AANT1, slr1737, IDI, and an antisenseconstruct for homogentisic acid dioxygenase.

[0423] The tocopherol composition and level in each plant generated bythe crosses (including all intermediate crosses) is also analyzed.Progeny of the transformants from these constructs will be crossed witheach other to stack the additional genes to reach the desired level oftocopherol.

[0424] A second set of DNA constructs is generated and referred to asthe “multiple gene constructs.” The multiple gene constructs containmultiple genes each under the control of a seed specific promoter suchas the arcelin 5, 7S α or napin promoter (Kridl et al., Seed Sci. Res.1:209:219 (1991) (Keegstra, Cell 56(2):247-53 (1989); Nawrath, et al.,Proc. Natl. Acad. Sci. U.S.A. 91:12760-12764 (1994)) and the geneproducts of each of the genes are targeted to the plastid by an encodedplastid target peptide. The multiple gene construct can have two or moreof the following genes: tyrA, slr1736, or ATPT2, dxs, dxr, GGPPS, HPPD,GMT, MT1, AANT1, slr1737, or its plant ortholog, IDI, and an antisenseconstruct for homogentisic acid dioxygenase.

[0425] Each construct is then transformed into at least one canola,Brassica napus or soybean plant. The tocopherol composition and level ineach plant is also analyzed using the method set forth in example 6.Progeny of the transformants from these constructs are crossed with eachother to stack the additional genes to reach the desired level oftocopherol.

EXAMPLE 8

[0426] Expression of the Anabaena MT1 coding sequence in Arabidopsis iscarried out. The Anabaena putative-MT1 coding sequence is amplified fromgenomic DNA derived from 3-day old Anabaena sp. (ATCC 27893) cultures.To isolate DNA, cultures are spun and the pellet washed with 1 ml PBS toremove media. Subsequently, the suspension is centrifuged and thesupernatant is discarded. The resulting pellet is resuspended in 1 ml ofwater and is boiled for 10 minutes. Anabaena DNA amplification reactionscontain 10 μL boiled Anabaena extract, the Expand™ High Fidelity PCRSystem and the oligonucleotide primers: 5′GGG GAC AAG TTT GTA CAA AAAAGC AGG CTT AGA AGG AGA TAG AAC CAT GAG TTG GTT GTT TTC TAC ACT GG 3′(SEQ ID NO: 83) and 5′GGG GAC CAC TTT GTA CAA GAA AGC TGG GTC CTA TTACTT TTG AGC AAC CTT GAT CG3′ (SEQ ID NO: 84). The reaction mix ispre-incubated for 5 minutes at 95° C., during which time the polymeraseis spiked in. The product is then amplified for 15 cycles of 94° C. for30 sec, 60° C. for 30 sec, and 72° C. for 1.5 minutes each. During thecycling, the annealing temperature is decreased by 1° C. per cycle foreach of the 15 cycles. An additional 15 cycles follow, consisting of 94°C. for 30 seconds, 45° C. for 30 seconds, and 72° C. for 1.5 minute,each followed by a 7 minute hold at 72° C.

[0427] After amplification, PCR products are purified using a Qiagen PCRcleanup column (Qiagen Company, Valencia, Calif.) and subcloned intopDONR™201 using the GATEWAY cloning system (Life Technologies,Rockville, Md.) to generate pMON67517. Sequences are confirmed by DNAsequencing using standard methodologies and then cloned into the napincassette derived from pCGN3223 (Kridl et al., Seed Sci. Res. 1:209-219(1991)) in a GATEWAY compatible binary destination vector containing theBAR selectable marker under the control of the 35S promoter. The MT1gene is cloned in as a translational fusion with the encoded plastidtarget peptide CTP1 (WO 00/61771) to target this protein to the plastidfrom pMON16600. The resultant expression vector (pMON67211) iselectroporated into ABI strain Agrobacterium cells and grown understandard conditions (McBride et al., Proc. Natl. Acad. Sci. USA91:7301-7305 (1994)) and vector fidelity is reconfirmed by restrictionanalysis. Transformation of pMON67211 into wild-type Arabidopsis,accession Columbia, as well as three high δ-tocopherol mutant lines(hdt2, hdt10, hdt16) is accomplished using the dipping method (Cloughand Bent, Plant J. 16(6):735-43 (1998)) and T₀ plants are grown in agrowth chamber under 16 h light, 19° C. T₁ seeds are sprinkled directlyonto soil, vernalized at 4° C. in the absence of light for 4 days, thentransferred to 21° C., 16 hours light. Transgenic plants are selected byspraying with a 1:200 dilution of Finale (AgrEvo Environmental Health,Montvale, N.J.) at 7 days and 14 days after seeding. Transformed plantsare grown to maturity and the T₂ seed is analyzed for tocopherol contentusing normal phase HPLC (Savidge, B. et al., Plant Physiology129:321-332 (2002)).

[0428] Two lines of pMON67211 in the hdt2 mutant line (67211-6 and67211-12) are taken forward to the next generation for examination ofphenotype in T₃ seed. In doing so, T₂ seeds are sprinkled directly ontosoil, vernalized at 4° C. in the absence of light for 4 days, thentransferred to 21° C., 16 hours light. Transgenic plants are selected byspraying with a 1:200 dilution of Finale (AgrEvo Environmental Health,Montvale, N.J.) at 7 and 14 days after seeding. Transformed plants aregrown to maturity (9 plants from line 6, 9 plants from line 12, and 4hdt2 mutant controls in one flat) and the T₃ seed is analyzed fortocopherol content using normal phase HPLC.

[0429]FIG. 25 shows the percent of seed δ-tocopherol in Arabidopsis T2seed from lines expressing MT 1 under the control of the napin promoter.

[0430] Table 8 below represents various data resulting from the abovetransformants. TABLE 8 Alpha strategy R₂ Arabidopsis seed: CTP-MT1 HPLCsequence and data folder SR022602 Sam- Sample Ng α ng γ ng δ ng totalAvg. ple wt. toco./mg toco./mg toco./mg toco./mg % Delta Name (mg) seedseed seed seed Serial Number Pedigree Gen Delta % 77 14 3.42 459.7220.17 483.31  9979-AT00002-54: @.0008. 9979 For 67211s 4.2 4.1 78 152.59 461.87 19.26 483.73  9979-AT00002-54: @.0009. 9979 For 67211s 4.089 13 4.78 459.21 16.34 480.33 AT_G193: @. PMON67211 T2 3.4 3.6 90 145.50 475.59 17.27 498.35 AT_G194: @. PMON67211 T2 3.5 86 13 5.64 476.7018.13 500.46 AT_G190: @. PMON67211 T2 3.6 82 13 6.19 476.84 18.10 501.13AT_G186: @. PMON67211 T2 3.6 88 14 7.13 477.51 19.27 503.91 AT_G192: @.PMON67211 T2 3.8 95 13 6.45 478.90 18.78 504.13 AT_G199: @. PMON67211 T23.7 85 11 5.67 480.31 19.62 505.60 AT_G189: @. PMON67211 T2 3.9 96 1310.08 480.68 18.69 509.45 AT_G200: @. PMON67211 T2 3.7 84 13 6.34 487.2318.47 512.04 AT_G188: @. PMON67211 T2 3.6 91 12 7.18 487.68 19.42 514.28AT_G195: @. PMON67211 T2 3.8 87 14 4.45 492.16 19.92 516.52 AT_G191: @.PMON67211 T2 3.9 93 13 7.07 492.17 18.19 517.43 AT_G197: @. PMON67211 T23.5 92 13 7.12 493.27 19.77 520.15 AT_G196: @. PMON67211 T2 3.8 94 138.28 494.79 18.04 521.11 AT_G198: @. PMON67211 T2 3.5 80 13 8.70 498.9418.71 526.36 AT_G184: @. PMON67211 T2 3.6 83 14 6.49 502.75 18.16 527.40AT_G187: @. PMON67211 T2 3.4 81 12 6.75 505.87 18.84 531.45 AT_G185: @.PMON67211 T2 3.5 9 12 3.66 277.61 265.61 546.88 hdt2: 0001. M5 48.6 48.110 10 5.62 268.82 239.24 513.69 hdt2: 0002. M5 46.6 11 13 4.80 266.70250.79 522.29 hdt2: 0003. M5 48.0 12 12 6.34 281.87 271.70 559.90 hdt2:0004. M5 48.5 13 12 4.75 277.59 266.87 549.21 hdt2: 0005. M5 48.6 18 134.38 410.93 146.44 561.74 67211-HDT2: 0005. T2 26.1 18.9 20 12 5.53421.63 133.57 560.73 67211-HDT2: 0007. T2 23.8 22 11 4.39 413.42 116.94534.75 67211-HDT2: 0009. T2 21.9 17 12 5.31 425.83 114.16 545.3067211-HDT2: 0004. T2 20.9 15 12 4.97 402.64 105.62 513.23 67211-HDT2:0002. T2 20.6 27 13 4.74 434.37 112.96 552.07 67211-HDT2: 0014. T2 20.516 13 5.98 416.73 108.13 530.84 67211-HDT2: 0003. T2 20.4 14 12 7.07431.05 107.70 545.81 67211-HDT2: 0001. T2 19.7 23 10 4.74 436.59 106.91548.24 67211-HDT2: 0010. T2 19.5 26 12 6.89 424.31 104.39 535.5967211-HDT2: 0013. T2 19.5 21 11 4.91 441.50 104.57 550.98 67211-HDT2:0008. T2 19.0 28 12 4.40 493.29 87.63 585.32 67211-HDT2: 0015. T2 15.024 13 4.20 452.86 74.83 531.89 67211-HDT2: 0011. T2 14.1 25 13 5.20510.41 72.70 588.31 67211-HDT2: 0012. T2 12.4 19 11 5.58 545.61 67.86619.05 67211-HDT2: 0006. T2 11.0 3 12.5 3.36 262.76 180.18 446.30 hdt16:@.0007. Control M5 40.4 38.2 2 9.6 2.54 305.52 178.20 486.25 hdt16:@.0005. Control M5 36.6 1 11.9 3.36 290.12 177.76 471.24 hdt16: @.0003.Control M5 37.7 11 10.1 2.02 255.50 169.29 426.81 AT_G58: @. PMON67211T2 39.7 15.3 12 12.4 5.28 352.67 100.76 458.71 AT_G59: @. PMON67211 T222.0 24 12.5 3.60 392.97 78.20 474.77 AT_G71: @. PMON67211 T2 16.5 14 123.90 380.29 72.98 457.18 AT_G61: @. PMON67211 T2 16.0 22 12.6 2.06370.66 68.50 441.22 AT_G69: @. PMON67211 T2 15.5 18 12.2 3.52 379.3870.29 453.19 AT_G65: @. PMON67211 T2 15.5 15 13 5.67 386.12 71.61 463.39AT_G62: @. PMON67211 T2 15.5 21 11.3 3.86 405.98 74.54 484.39 AT_G68: @.PMON67211 T2 15.4 25 12.6 6.42 408.38 74.56 489.36 AT_G72: @. PMON67211T2 15.2 19 12.5 3.95 412.64 72.24 488.84 AT_G66: @. PMON67211 T2 14.8 2012.7 2.99 431.01 65.65 499.66 AT_G67: @. PMON67211 T2 13.1 17 12.3 5.77423.19 48.73 477.70 AT_G64: @. PMON67211 T2 10.2 23 11.3 2.35 408.2445.41 456.00 AT_G70: @. PMON67211 T2 10.0 10 11.9 7.81 443.06 43.58494.45 AT_G57: @. PMON67211 T2 8.8 13 12.6 3.64 421.06 38.53 463.23AT_G60: @. PMON67211 T2 8.3 16 12.9 3.76 430.69 37.10 471.56 AT_G63: @.PMON67211 T2 7.9 33 13.2 4.32 356.41 71.85 432.59 hdt10: @.0001. ControlM6 16.6 9.6 34 13.1 5.73 469.11 12.79 487.62 hdt10: @.0002. Control M62.6 56 13.3 4.77 361.67 63.37 429.82 AT_G48: @. PMON67211 T2 14.7 4.7 618.1 2.70 351.84 50.96 405.50 AT_G54: @. PMON67211 T2 12.6 54 12.2 5.66432.55 41.60 479.81 AT_G46: @. PMON67211 T2 8.7 59 13.9 5.18 416.8838.34 460.40 AT_G52: @. PMON67211 T2 8.3 51 13 3.99 430.18 22.41 456.58AT_G43: @. PMON67211 T2 4.9 58 12.2 4.88 463.37 21.72 489.97 AT_G51: @.PMON67211 T2 4.4 52 13.4 5.34 442.72 18.24 466.31 AT_G44: @. PMON67211T2 3.9 64 12.6 5.50 477.62 10.72 493.84 AT_G117: @. PMON67211 T2 2.2 5712.7 6.27 467.48 9.12 482.88 AT_G50: @. PMON67211 T2 1.9 50 13.1 4.83450.16 7.94 462.93 AT_G42: @. PMON67211 T2 1.7 63 12.8 4.78 445.42 7.81458.00 AT_G56: @. PMON67211 T2 1.7 55 12.6 8.32 460.07 7.58 475.98AT_G47: @. PMON67211 T2 1.6 53 13.3 6.43 417.71 6.76 430.91 AT_G45: @.PMON67211 T2 1.6 62 12.6 5.36 473.04 6.88 485.28 AT_G55: @. PMON67211 T21.4 60 12.9 4.87 463.45 5.68 474.00 AT_G53: @. PMON67211 T2 1.2

[0431]FIG. 26 shows T₃ seed δ-tocopherol percentage from two linesexpressing. MT1 under the control of the napin promoter (pMON6721 1).Table 9 below shows T₃ seed data from hdt2 mutant lines transformed withpMON67211. TABLE 9 Crop Biotype Pedigree mp: aT mp: gT mp: dT totaltoco. % delta Gen AT SEED hdt2: @.0001.0001. 2 280 190 472 40.3 M7 ATSEED hdt2: @.0001.0003. 4 263 204 471 43.3 M7 AT SEED hdt2: @.0001.0002.3 262 208 473 44.0 M7 AT SEED hdt2: @.0001.0004. 4 271 220 495 44.4 M767211-6 11.0 R2 AT SEED 67211-HDT2: 0006.0005. 4 398 83 485 17.1 R3 ATSEED 67211-HDT2: 0006.0001. 3 438 60 501 12.0 R3 AT SEED 67211-HDT2:0006.0008. 4 453 59 516 11.4 R3 AT SEED 67211-HDT2: 0006.0002. 3 448 56507 11.0 R3 AT SEED 67211-HDT2: 0006.0004. 2 417 52 471 11.0 R3 AT SEED67211-HDT2: 0006.0007. 3 468 50 521 9.6 R3 AT SEED 67211-HDT2:0006.0006. 4 464 45 513 8.8 R3 AT SEED 67211-HDT2: 0006.0009. 5 456 42503 8.3 R3 AT SEED 67211-HDT2: 0006.0003. 4 456 30 490 6.1 R3 67211-1212.4 R2 AT SEED 67211-HDT2: 0012.0002. 4 373 102 479 21.3 R3 AT SEED67211-HDT2: 0012.0009. 3 399 98 500 19.6 R3 AT SEED 67211-HDT2:0012.0003. 3 397 92 492 18.7 R3 AT SEED 67211-HDT2: 0012.0001. 4 440 66510 12.9 R3 AT SEED 67211-HDT2: 0012.0008. 2 469 65 536 12.1 R3 AT SEED67211-HDT2: 0012.0006. 4 438 53 495 10.7 R3 AT SEED 67211-HDT2:0012.0004. 5 465 54 524 10.3 R3 AT SEED 67211-HDT2: 0012.0005. 5 460 52517 10.1 R3 AT SEED 67211-HDT2: 0012.0007. 3 458 47 508 9.3 R3

EXAMPLE 9

[0432] The CTP-MT1 gene described in example 8 is cloned behind thenapin promoter into a binary vector with the ATPT2 gene from Arabidopsisand in another double construct with the prenyltransferase (PT) gene(SLR1736 ORF) from Synechocystis (described in PCT application WO0063391).

[0433] The MT1 gene is cut out of vector pMON67517 using the restrictionenzymes BspHI/PstI and cloned into the PstI/NcoI digested vectorbackbone of the napin shuttle vector pMON16600, resulting in theformation of pMON67210. The napin cassette from pMON67210, containingthe MT1 gene as a translational fusion with the encoded plastid targetpeptide CTP1 (WO 00/61771) is then cut from this vector with Not I andthe ends filled in with dNTPs using a Klenow procedure. The resultingfragment is inserted into vectors pMON16602 (digested with PmeI) andpCGN10822 (digested with SnaBI) to make pMON67213 and pMON67212,respectively (FIGS. 27 and 28). Vectors pMON16602 and pCGN10822 aredescribed in PCT application WO 0063391.

[0434] These double constructs express the MT1 gene and thehomogentisate prenyl-transferase from either Arabidopsis orSynechocystis under the control of the napin seed-specific promoter. Thedouble gene constructs are used to transform Arabidopsis and transformedplants are grown to maturity as detailed in Example 2. The resulting T₂seed is analyzed for total tocopherol content and composition usinganalytical procedures described in Example 2. FIGS. 29-32 show total,γ-, δ-, and α-tocopherol levels for various transformed plant lines.Table 10 provides further data from the above-described transformations.TABLE 10 ng α ng γ ng δ ng total toco./mg toco./mg toco./mg toco./mgseed seed seed seed serial number Pedigree Construct 6.28 520.72 13.30540.30 69000157657 AT00002: @.0321. Control For 67212s 5.83 612.04 10.36628.24 69000157645 AT00002: @.0322. Control For 67212s 7.34 621.17 12.62641.14 69000157633 AT00002: @.0323. Control For 67212s 6.48 609.23 13.41629.12 69000157621 AT00002: @.0324. Control For 67212s 6.28 421.10 9.19436.56 69000157710 AT_G73: @. PMON67212 4.72 433.54 7.99 446.2469000157746 AT_G76: @. PMON67212 7.83 570.77 8.77 587.37 69000157758AT_G77: @. PMON67212 7.38 588.65 8.70 604.74 69000157784 AT_G80: @.PMON67212 9.56 580.79 14.93 605.28 69000157722 AT_G74: @. PMON67212 5.99605.44 10.38 621.82 69000157847 AT_G86: @. PMON67212 7.66 615.03 12.84635.53 69000157859 AT_G87: @. PMON67212 8.29 634.10 9.58 651.9769000157734 AT_G75: @. PMON67212 8.82 628.29 15.95 653.06 69000157809AT_G82: @. PMON67212 7.41 636.96 10.07 654.45 69000157823 AT_G84: @.PMON67212 6.64 648.21 10.25 665.10 69000157861 AT_G88: @. PMON67212 7.46624.59 34.85 666.91 69000157811 AT_G83: @. PMON67212 8.07 668.83 11.37688.27 69000157760 AT_G78: @. PMON67212 7.96 691.84 11.38 711.1869000157835 AT_G85: @. PMON67212 7.26 705.18 12.01 724.44 69000157796AT_G81: @. PMON67212 7.95 708.29 12.64 728.88 69000157772 AT_G79: @.PMON67212 6.95 508.05 11.25 526.25 69000157582 AT00002: @.0328. ControlFor 67213s 8.16 513.84 14.12 536.11 69000157619 AT00002: @.0325. ControlFor 67213s 8.94 547.41 16.60 572.95 69000157607 AT00002: @.0326. ControlFor 67213s 7.83 483.85 15.95 507.63 69000157974 AT_G99: @. PMON672138.50 488.67 15.92 513.09 69000157671 AT_G101: @. PMON67213 7.18 503.5013.74 524.42 69000157873 AT_G89: @. PMON67213 6.31 511.87 15.83 534.0169000157950 AT_G97: @. PMON67213 7.30 515.26 11.47 534.02 69000157897AT_G91: @. PMON67213 7.11 512.25 19.56 538.92 69000157962 AT_G98: @.PMON67213 6.61 525.17 12.82 544.60 69000157900 AT_G92: @. PMON67213 7.50521.38 16.85 545.73 69000157683 AT_G102: @. PMON67213 7.87 529.25 11.29548.41 69000157948 AT_G96: @. PMON67213 6.88 523.01 18.83 548.7269000157912 AT_G93: @. PMON67213 7.56 534.21 13.03 554.80 69000157669AT_G100: @. PMON67213 6.79 536.89 12.17 555.86 69000157885 AT_G90: @.PMON67213 7.83 535.00 17.97 560.80 69000157936 AT_G95: @. PMON67213 8.57532.53 21.13 562.23 69000157708 AT_G104: @. PMON67213 8.15 550.66 18.42577.23 69000157695 AT_G103: @. PMON67213 9.91 560.45 26.66 597.0269000157924 AT_G94: @. PMON67213

[0435]

1 85 1 1047 DNA Arabidopsis thaliana 1 atgaaagcaa ctctagcagc accctcttctctcacaagcc tcccttatcg aaccaactct 60 tctttcggct caaagtcatc gcttctctttcggtctccat cctcctcctc ctcagtctct 120 atgacgacaa cgcgtggaaa cgtggctgtggcggctgctg ctacatccac tgaggcgcta 180 agaaaaggaa tagcggagtt ctacaatgaaacttcgggtt tgtgggaaga gatttgggga 240 gatcatatgc atcatggctt ttatgaccctgattcttctg ttcaactttc tgattctggt 300 cacaaggaag ctcagatccg tatgattgaagagtctctcc gtttcgccgg tgttactgat 360 gaagaggagg agaaaaagat aaagaaagtagtggatgttg ggtgtgggat tggaggaagc 420 tcaagatatc ttgcctctaa atttggagctgaatgcattg gcattactct cagccctgtt 480 caggccaaga gagccaatga tctcgcggctgctcaatcac tctctcataa ggcttccttc 540 caagttgcgg atgcgttgga tcagccattcgaagatggaa aattcgatct agtgtggtcg 600 atggagagtg gtgagcatat gcctgacaaggccaagtttg taaaagagtt ggtacgtgtg 660 gcggctccag gaggtaggat aataatagtgacatggtgcc atagaaatct atctgcgggg 720 gaggaagctt tgcagccgtg ggagcaaaacatcttggaca aaatctgtaa gacgttctat 780 ctcccggctt ggtgctccac cgatgattatgtcaacttgc ttcaatccca ttctctccag 840 gatattaagt gtgcggattg gtcagagaacgtagctcctt tctggcctgc ggttatacgg 900 actgcattaa catggaaggg ccttgtgtctctgcttcgta gtggtatgaa aagtattaaa 960 ggagcattga caatgccatt gatgattgaaggttacaaga aaggtgtcat taagtttggt 1020 atcatcactt gccagaagcc actctaa 10472 1047 DNA Arabidopsis thaliana 2 atgaaagcaa ctctagcagc accctcttctctcacaagcc tcccttatcg aaccaactct 60 tctttcggct caaagtcatc gcttctctttcggtctccat cctcctcctc ctcagtctct 120 atgacgacaa cgcgtggaaa cgtggctgtggcggctgctg ctacatccac tgaggcgcta 180 agaaaaggaa tagcggagtt ctacaatgaaacttcgggtt tgtgggaaga gatttgggga 240 gatcatatgc atcatggctt ttatgaccctgattcttctg ttcaactttc tgattctggt 300 cacaaggaag ctcagatccg tatgattgaagagtctctcc gttttgccgg tgttactgat 360 gaagaggagg agaaaaagat aaagaaagtagtggatgttg ggtgtgggat tggaggaagc 420 tcaagatatc ttgcctctaa atttggagctgaatgcattg gcattactct cagccctgtt 480 caggccaaga gagccaatga tctcgcggctgctcaatcac tcgctcataa ggcttccttc 540 caagttgcgg atgcgttgga tcagccattcgaagatggaa aattcgatct agtgtggtcg 600 atggagagtg gtgagcatat gcctgacaaggccaagtttg taaaagagtt ggtacgtgtg 660 gcggctccag gaggtaggat aataatagtgacatggtgcc atagaaatct atctgcgggg 720 gaggaagctt tgcagccgtg ggagcaaaacatcttggaca aaatctgtaa gacgttctat 780 ctcccggctt ggtgctccac cgatgattatgtcaacttgc ttcaatccca ttctctccag 840 gatattaagt gtgcggattg gtcagagaacgtagctcctt tctggcctgc ggttatacgg 900 actgcattaa catggaaggg ccttgtgtctctgcttcgta gtggtatgaa aagtattaaa 960 ggagcattga caatgccatt gatgattgaaggttacaaga aaggtgtcat taagtttggt 1020 atcatcactt gccagaagcc actctaa 10473 1095 DNA Oryza sativa 3 atggcccacg ccgccgcggc cacgggcgca ctggcaccgctgcatccact gctccgctgc 60 acgagccgtc atctctgcgc ctcggcttcc cctcgcgccggcctctgcct ccaccaccac 120 cgccgccgcc gccgcagcag ccggaggacg aaactcgccgtgcgcgcgat ggcaccgacg 180 ttgtcctcgt cgtcgacggc ggcggcagct cccccggggctgaaggaggg catcgcgggg 240 ctctacgacg agtcgtccgg cgtgtgggag agcatctggggcgagcacat gcaccacggc 300 ttctacgacg ccggcgaggc cgcctccatg tccgaccaccgccgcgccca gatccgcatg 360 atcgaggaat ccctcgcctt cgccgccgtc cccggtgcagatgatgcgga gaagaaaccc 420 aaaagtgtag ttgatgttgg ctgtggcatt ggtggtagctcaagatactt ggcgaacaaa 480 tacggagcgc aatgctacgg catcacgttg agtccggtgcaggctgaaag aggaaatgcc 540 ctcgcggcag agcaagggtt atcagacaag gtgcgtattcaagttggtga tgcattggag 600 cagccttttc ctgatgggca gtttgatctt gtctggtccatggagagtgg cgagcacatg 660 ccagacaaac ggcagtttgt aagcgagctg gcacgcgtcgcagctcctgg ggcgagaata 720 atcattgtga cctggtgcca taggaacctc gagccatccgaagagtccct gaaacctgat 780 gagctgaatc tcctgaaaag gatatgcgat gcatattatctcccagactg gtgctctcct 840 tctgattatg tcaaaattgc cgagtcactg tctcttgaggatataaggac agctgattgg 900 tcagagaacg tcgccccatt ctggcctgcg gttataaaatcagcattgac atggaaaggt 960 ttaacttctc tgctaagaag tgggtggaag acgataagaggtgcaatggt gatgcctctg 1020 atgatcgaag gatacaagaa agggctcatc aaattcaccatcatcacctg tcgcaagccc 1080 gaaacaacgc agtag 1095 4 1038 DNA Gossypiumhirsutum 4 atggctgccg cgttacaatt acaaacacac ccttgcttcc atggcacgtgccaactctca 60 cctccgccac gaccttccgt ttccttccct tcttcctccc gctcgtttccatctagcaga 120 cgttccctgt ccgcgcatgt gaaggcggcg gcgtcgtctt tgtccaccaccaccttgcag 180 gaagggatag cggagtttta cgatgagtcg tcggggattt gggaagacatatggggtgac 240 catatgcacc atggatatta cgagccgggt tccgatattt cgggttcagatcatcgtgcc 300 gctcagattc gaatggtcga agaatcgctc cgttttgctg gaatatcagaggacccagca 360 aacaggccca agagaatagt tgatgttggg tgtgggatag gaggcagttctaggtatcta 420 gcaaggaaat atggggcaaa atgccaaggc attactttga gccctgttcaagctggaaga 480 gccaatgctc ttgctaatgc tcaaggacta gcagaacagg tttgttttgaagttgcagat 540 gccttgaacc aaccattccc tgatgaccaa tttgatcttg tttggtctatggaaagcgga 600 gaacacatgc ctgacaaacc caagtttgtt aaagagctgg tgcgagtggcagctccagga 660 ggcacaataa tagtagtgac atggtgccat agggatcttg gtccatctgaagagtctttg 720 cagccatggg agcaaaagct tttaaacaga atatgtgatg cttactatttaccagagtgg 780 tgttctactt ctgattatgt caaattattt cagtccctat ctctccaggatataaaggca 840 ggagactgga ctgagaatgt agcacccttt tggccagcag tgatacgttcagcattgaca 900 tggaagggct tcacatcgct gctacgaagt ggattaaaaa caataaaaggtgcactggtg 960 atgccattga tgatcgaagg tttccagaaa ggggtgataa agtttgccatcattgcttgc 1020 cggaagccag ctgagtag 1038 5 1131 DNA Cuphea pulcherrima 5atgccgataa catctatttc cgcaaaccaa aggccattct tcccctcacc ttatagaggc 60agctccaaga acatggcacc gcccgaactg gctcagtcgc aagtacctat gggaagtaac 120aagagcaaca agaaccacgg cttggtcggt tcggtttctg gttggagaag gatgtttggg 180acatgggcta ctgccgacaa gactcagagt accgatacgt ctaatgaagg cgtggttagt 240tacgatactc aggtcttgca gaagggtata gcggagttct atgacgagtc gtcgggtata 300tgggaggata tatggggaga tcacatgcat catggctact atgatggttc cactcctgtc 360tccctcccag accatcgctc tgcgcagatc cgaatgattg acgaggctct ccgctttgcc 420tcggttcctt caggagaaga agatgagtcc aagtctaaga ttccaaagag gatagtggat 480gtcgggtgtg ggataggggg aagctccaga tacctggcta gaaaatatgg cgccgagtgt 540cggggcatca ctctcagtcc tgtccaggct gagaggggca attcacttgc acggtctcaa 600ggtctttctg acaaggtctc ctttcaagtc gccgatgctt tggcacagcc atttcccgat 660ggacagtttg atttggtctg gtccatggag agcggggaac acatgcccga caagagcaag 720tttgtcaatg agctagtaag agtagcagct ccgggtggca cgataataat tgtcacatgg 780tgccatagag atctcaggga agacgaagat gcgctgcagc ctcgggagaa agagatattg 840gacaagatat gcaacccctt ttatcttccc gcctggtgtt ctgctgccga ctatgttaag 900ttgctccagt cacttgatgt cgaggacatt aaatctgcgg actggactcc atatgttgcc 960ccattttggc cagctgtgct gaagtccgct ttcactataa agggcttcgt gtctctattg 1020aggagcggaa tgaagaccat aaagggagca tttgcaatgc cgctgatgat cgaaggatac 1080aagaaaggtg tcatcaagtt ttccatcatc acatgccgta agcccgaata g 1131 6 2045 DNABrassica napus 6 atgaaagcga ctctcgcacc ctcctctctc ataagcctcc ccaggcacaaagtatcttct 60 ctccgttcac cgtcgcttct ccttcagtcc caacggccat cctcagccttaatgacgacg 120 acgacggcat cacgtggaag cgtggctgtg acggctgctg ctacctcctccgttgaggcg 180 ctgcgggaag gaatagcgga attctacaac gagacgtcgg gattatgggaggagatttgg 240 ggagatcata tgcatcacgg cttctacgat cctgattcct ctgttcaactttcagattcc 300 ggtcaccggg aagctcagat ccggatgatc gaagagtctc tacgtttcgccggcgttact 360 ggttcgcttc tcatgctata cagttagagt ttgattcgtt gtttgttatgaatgataaac 420 ctacacatga acactttcta gatttattat aaacattctt tttgaacttatattataaac 480 aattcttaca aacaaaatgc tctttgaact cttaaaaata tataacaatggtttagtttt 540 gatttgtcgg taagagaaat gagtagggat gtttgaagcc agataaagcctttcttttat 600 ccctggggag aggcttacag taagccacgt cccatccaga agcagacccattccctaact 660 aggctggatg atgataaata agttcttcct catttcaaga ttaagaaaacaatctaaact 720 gaaataataa cgcgcagtcg gtgaaaatat ctttatgctt gggattgttgttgttattat 780 taatttatat tataaacaca tgaccttttt aaagaagagg agaaaaagataaagagagta 840 gtggatgttg ggtgtgggat cggcggaagc tcaaggtata ttgcctctaaatttggtgcc 900 gaatgcattg gcatcacact cagtcccgtt caagccaaga gagccaatgatctcgccgcc 960 gctcaatcac tctctcataa ggtgtcttct tgtacattcg accatttttttctgcggaat 1020 ctgagctaac tgagacgcca ctggaccagg tttccttcca agttgcagatgcactggagc 1080 aaccatttga agatggtata ttcgatcttg tgtggtcaat ggaaagcggtgagcatatgc 1140 ctgacaaggc caaggtatac tacctagctc accataatct ttatactagatttagtagac 1200 aatatccatc ttttggatgt caatgatgtc cattaatttt taaataaacaaaataaaaaa 1260 tgagagtaaa attttttttt gtcaaactta tctaataaat attatgtaataataccacgt 1320 ttttctattt aattatggca tggtttcttt tttttttgtc taaaaaaaattgtagtatct 1380 gttagaaaac agaatctaag tatgatattt ttgaaactca ttcagtcttcgttgtggaag 1440 tatatttacc gtgtgtgcga aatgagtgta gttcgtgaag gaattggtacgtgtggcggc 1500 tccaggagga aggataataa tagtgacatg gtgccacaga aatctatctccaggggaaga 1560 ggctttgcag ccatgggagc agaacctctt ggacagaatc tgcaaaacattttatctccc 1620 agcctggtgc tccacctcgg attatgtcga tttgcttcag tccctctcgctccaggttat 1680 tatatttctc acgctccaat tgctaaaatt agtacttgga gctagttaagtagtgtctca 1740 aatatatgtg tgtttgtagg atattaagtg tgcagattgg tcagagaacgtagctccttt 1800 ctggccggcg gttatacgaa ccgcattaac gtggaagggc cttgtgtctctgcttcgtag 1860 tggtatgttt ccgcaatgtt gttcacattc atgattttta taagattagaactaaggttg 1920 ttgggtgtcg gaaacgcaca ggtatgaaga gtataaaagg agcattgacaatgccattga 1980 tgattgaagg gtacaagaaa ggtgtcatta agtttggcat catcacttgccagaagcctc 2040 tctaa 2045 7 2973 DNA Brassica napus 7 atgaaagcgacactcgcacc accctcctct ctcataagcc tccccaggca caaagtatct 60 tccctccgttcaccgtcgct tctccttcag tcccaacggc gatcctcagc cttaatgacg 120 acgacggcatcacgtggaag cgtggctgtg acggctgctg ctacctcctc cgctgaggcg 180 ctgcgagaaggaatagcgga attctacaac gagacgtcgg gattatggga ggagatttgg 240 ggagatcatatgcatcacgg cttctacgat cccgattcct ctgttcaact ttcagattcc 300 ggtcaccgggaagctcagat ccggatgatt gaagagtctc tacgtttcgc cggcgttact 360 ggttcgcttctcatgctcta cacttgagtt tgatacgttg tttattataa acattttttt 420 gaacttttattataaacaat tcttacaaac aaattactct ttgaactctt taaaatctat 480 aacaaaggtttagttttact ttttatttgt tgttggtaac agaaatgagt agggatgttt 540 gaagtcagatatagcctttc tgtttatccc ttgggaagaa aggcttacag taagccacgt 600 cccatccagaagcagaccca ttccctaact aatcattttt atgaacaatt tgtaacacta 660 ttattcctagatattttttt tttacgttta gttaccctaa ctctttgtat ataagacaag 720 aggtgatttttcacattata tatcaaaaca tagacatagt ttttttgaga aaatatatca 780 tacatagttgtaacttagaa ttatatattt ttgagaaaaa aactcagtaa taattttctt 840 ataattattcatagttttat atttattaat aataagattt tgtaagctct ttttgaaact 900 attatggataatgaataagt tccccatttc aagattaaga aaacaattta aactgaaata 960 ataatgcgcattcggtgaaa atatctttct gcttgggatt gttgttgtta atctatatta 1020 ttaaaactgaagtacatttt ggtactgttt ggaaacttag atagtagatt aaatgaaaat 1080 tgtttggaaacaaggatagc agattaaata tttttttatt tacatattta gtcactgtat 1140 ttctttctcatttacagatt ctgtcgtttg gaaacttgga tagcagatta aatgaaaaat 1200 gtttggaaacacagttaaca tattaaatat ctatttttat ttcatattta gccattgcat 1260 ttctttcttatttacaaatc tgccacttca cttaaaataa aaaaattaaa ttaattacaa 1320 tgaattgttatttctttttg ctgaaaataa aaacgcaaac tgcaatatat agtatatatt 1380 aatctgctacaatacaattt tcaagaaaac caaatatcat aaaattaata ataatttata 1440 aaaacctacagtaaaaaaat aaatcatttt taaataaata aacaaaaaaa atcaataggt 1500 tgatatatgaatattacaat tacatcaaat tgcatcaagt tataaaatta taaatataat 1560 attacgtacaaataaaaatt attatcaaac atctatttta taatataata tattctactc 1620 taaatatatttacaaaacat aaaaatataa atggacattt tataaaatca atggtttata 1680 agtttacattgaacgcaagt taaattccaa catccgcgcg gggcgcgggt caagatctag 1740 tattaatttatattataaac acatgacttt ttttaaagaa gaggagaaaa agataaagag 1800 agtggtggatgttgggtgtg ggatcggagg aagctcaagg tatattgcct ctaaatttgg 1860 tgccgaatgcattggcatca cactcagtcc cgttcaagcc aagagagcaa atgatctcgc 1920 caccgctcaatcactctctc ataaggtgtc ttctcgtaca ttcgaccatt ctttctgcgg 1980 ataatctgatctaactgaga cgccattgga ccaggtttcc ttccaagttg cagatgcatt 2040 ggaccaaccatttgaagatg gtatatccga tcttgtttgg tcaatggaaa gcggtgagca 2100 tatgcctgacaaggccaagg tatactagct cagcataact tttatactag atttactaga 2160 caatatctatcttttcatgt caatgatgtc caataatttt aaaataaaca aaagaaggat 2220 gtgagggtaaaattttgtca aatttatata acaacacgtt ttctatttag ttatgtcatg 2280 gtttctttttgtctaaaaaa ttttaggcag agtttacaaa aagaaaattg tagtatctgt 2340 tcgaaaacagaatcttagtg tggtatttta gaaactcatt cagtcttcct tgtggaagca 2400 tatttactgtgtgtgcgaaa tgagtgtagt tcgtgaagga attggtacgt gtgacggctc 2460 caggaggaaggataataata gtgacatggt gccacagaaa tctatctcaa ggggaagaat 2520 ctttgcagccatgggagcag aacctcttgg acagaatctg caaaacattt tatctcccgg 2580 cctggtgctccaccactgat tatgtcgagt tgcttcaatc cctctcgctc caggttatta 2640 tatttctcacgctccgatgc taaaatcagt aagtattgtc tcaaatatat gtgtgtttgt 2700 aggatattaagtatgcagat tggtcagaga acgtagctcc tttctggccg gcggttatac 2760 gaaccgcattaacgtggaag ggccttgtgt ctctgcttcg tagtggtatg tttccgcaat 2820 gttgtttacattcatgattc caaatgttta taagattaga aacatacagg tatgaagagt 2880 ataaaaggagcattgacaat gccattgatg attgaagggt acaagaaagg tgtcattaag 2940 tttggcatcatcacttgcca gaagcctcta taa 2973 8 1044 DNA Brassica napus 8 atgaaagcgactctcgcacc ctcctctctc ataagcctcc ccaggcacaa agtatcttct 60 ctccgttcaccgtcgcttct ccttcagtcc caacggccat cctcagcctt aatgacgacg 120 acgacggcatcacgtggaag cgtggctgtg acggctgctg ctacctcctc cgttgaggcg 180 ctgcgggaaggaatagcgga attctacaac gagacgtcgg gattatggga ggagatttgg 240 ggagatcatatgcatcacgg cttctacgat cctgattcct ctgttcaact ttcagattcc 300 ggtcaccgggaagctcagat ccggatgatc gaagagtctc tacgtttcgc cggcgttact 360 gaagaggagaaaaagataaa gagagtagtg gatgttgggt gtgggatcgg cggaagctca 420 aggtatattgcctctaaatt tggtgccgaa tgcattggca tcacactcag tcccgttcaa 480 gccaagagagccaatgatct cgccgccgct caatcactct ctcataaggt ttccttccaa 540 gttgcagatgcactggagca accatttgaa gatggtatat tcgatcttgt gtggtcaatg 600 gaaagcggtgagcatatgcc tgacaaggcc aagttcgtga aggaattggt acgtgtggcg 660 gctccaggaggaaggataat aatagtgaca tggtgccaca gaaatctatc tccaggggaa 720 gaggctttgcagccatggga gcagaacctc ttggacagaa tctgcaaaac attttatctc 780 ccagcctggtgctccacctc ggattatgtc gatttgcttc agtccctctc gctccaggat 840 attaagtgtgcagattggtc agagaacgta gctcctttct ggccggcggt tatacgaacc 900 gcattaacgtggaagggcct tgtgtctctg cttcgtagtg gtatgaagag tataaaagga 960 gcattgacaatgccattgat gattgaaggg tacaagaaag gtgtcattaa gtttggcatc 1020 atcacttgccagaagcctct ctaa 1044 9 1044 DNA Brassica napus 9 atgaaagcga cactcgcaccaccctcctct ctcataagcc tccccaggca caaagtatct 60 tccctccgtt caccgtcgcttctccttcag tcccaacggc gatcctcagc cttaatgacg 120 acgacggcat cacgtggaagcgtggctgtg acggctgctg ctacctcctc cgctgaggcg 180 ctgcgagaag gaatagcggaattctacaac gagacgtcgg gattatggga ggagatttgg 240 ggagatcata tgcatcacggcttctacgat cccgattcct ctgttcaact ttcagattcc 300 ggtcaccggg aagctcagatccggatgatt gaagagtctc tacgtttcgc cggcgttact 360 gaagaggaga aaaagataaagagagtggtg gatgttgggt gtgggatcgg aggaagctca 420 aggtatattg cctctaaatttggtgccgaa tgcattggca tcacactcag tcccgttcaa 480 gccaagagag caaatgatctcgccaccgct caatcactct ctcataaggt ttccttccaa 540 gttgcagatg cattggaccaaccatttgaa gatggtatat ccgatcttgt ttggtcaatg 600 gaaagcggtg agcatatgcctgacaaggcc aagttcgtga aggaattggt acgtgtgacg 660 gctccaggag gaaggataataatagtgaca tggtgccaca gaaatctatc tcaaggggaa 720 gaatctttgc agccatgggagcagaacctc ttggacagaa tctgcaaaac attttatctc 780 ccggcctggt gctccaccactgattatgtc gagttgcttc agtccctctc gctccaggat 840 attaagtatg cagattggtcagagaacgta gctcctttct ggccggcggt tatacgaacc 900 gcattaacgt ggaagggccttgtgtctctg cttcgtagtg gtatgaagag tataaaagga 960 gcattgacaa tgccattgatgattgaaggg tacaagaaag gtgtcattaa gtttggcatc 1020 atcacttgcc agaagcctctctaa 1044 10 933 DNA Lycopersicon esculentum 10 atggctagtg ttgctgcgatgaatgctgtg tcttcgtcat ctgtagaagt tggaatacag 60 aatcaacagg agctgaaaaaaggaattgca gatttatatg atgagtcttc tgggatttgg 120 gaagatattt ggggtgaccatatgcatcat ggatattatg aacctaaatc ctctgtggaa 180 ctttcagatc atcgtgctgctcagatccgt atgattgaac aggctctaag ttttgctgct 240 atttctgaag atccagcgaagaaaccaacg tccatagttg atgttggatg tggcatcggt 300 ggcagttcta ggtaccttgcaaagaaatat ggcgctacag ctaaaggtat cactttgagt 360 cctgtacaag cagagagggctcaagctctt gctgatgctc aaggattagg tgataaggtt 420 tcatttcaag tagcagacgccttgaatcag ccttttccag atgggcaatt cgacttggtt 480 tggtccatgg agagtggagaacacatgccg aacaaagaaa agtttgttgg cgaattagct 540 cgagtggcag caccaggaggcacaatcatc cttgtcacat ggtgccacag ggacctttcc 600 ccttcggagg aatctctgactccagaggag aaagagctgt taaataagat atgcaaagcc 660 ttctatcttc cggcttggtgttccactgct gattatgtga agttacttca atccaattct 720 cttcaggata tcaaggcagaagactggtct gagaatgttg ctccattttg gccagcagtc 780 ataaagtcag cactgacatggaagggcttc acatcagtac tacgcagtgg atggaagaca 840 atcaaagctg cactggcaatgccactgatg attgaaggat acaagaaagg tctcatcaaa 900 tttgccatca tcacatgtcgaaaacctgaa taa 933 11 909 DNA Glycine max 11 atgtcggtgg agcagaaagcagcagggaag gaggaggagg gaaaactgca gaagggaatt 60 gcagagttct acgacgagtcgtctggcata tgggagaaca tttggggcga tcacatgcac 120 cacggctttt atgacccggattccaccgtt tctgtttctg atcatcgcgc tgctcagatc 180 cgaatgatcc aagaatctcttcgttttgcc tctctgcttt ctgagaaccc ttctaaatgg 240 cccaagagta tagttgatgttgggtgtggc atagggggca gctccagata cctggccaag 300 aaatttggag caacgagcgtaggcattact ctgagtcctg ttcaagctca aagagcaaat 360 gctcttgctg ctgctcaaggattggctgat aaggtttcct ttcaggttgc tgacgctcta 420 cagcaaccat tctctgacggccagtttgat ctggtgtggt ccatggagag tggagagcat 480 atgcctgaca aagctaagtttgttggagag ttagctcggg tagcagcacc aggtgccact 540 ataataatag taacatggtgccacagggat cttggccctg acgaacaatc cttacatcca 600 tgggagcaag atctcttaaagaagatttgc gatgcatatt acctccctgc ctggtgctca 660 acttctgatt atgttaagttgctccaatcc ctgtcacttc aggacatcaa gtcagaagat 720 tggtctcgct ttgttgctccattttggcca gcagtgatac gctcagcctt cacatggaag 780 ggtctaactt cactcttgagcagtggacaa aaaacgataa aaggagcttt ggctatgcca 840 ttgatgatag agggatacaagaaagatcta attaagtttg ccatcattac atgtcgaaaa 900 cctgaataa 909 12 1053DNA Glycine max 12 atggccaccg tggtgaggat cccaacaatc tcatgcatccacatccacac gttccgttcc 60 caatcccctc gcactttcgc cagaatccgg gtcggacccaggtcgtgggc tcctattcgg 120 gcatcggcag cgagctcgga gagaggggag atagtattggagcagaagcc gaagaaggag 180 gaggagggga aactgcagaa gggaatcgca gagttctacgacgagtcgtc tggcttatgg 240 gagaacattt ggggcgacca catgcaccat ggcttttatgacccggattc cactgtttct 300 gtttctgatc atcgcgctgc tcagatccga atgatccaagagtctcttcg ctttgcctct 360 gtttctgagg agcgtagtaa atggcccaag agtatagttgatgttgggtg tggcataggt 420 ggcagctcca gatacctggc caagaaattt ggagcaaccagcgtaggcat tactctgagt 480 cctgttcaag ctcaaagagc aaatgctctt gctgctgctcaaggattggc tgataaggtt 540 tcctttcagg ttgctgacgc tctacagcaa ccattctctgacggccagtt tgatctggtg 600 tggtccatgg agagtggaga gcatatgcct gacaaagctaagtttgttgg agagttagct 660 cgggtagcag caccaggtgc cactataata atagtaacatggtgccacag ggatcttggc 720 cctgacgaac aatccttaca tccatgggag caagatctcttaaagaagat ttgcgatgca 780 tattaccttc ctgcctggtg ctcaacttct gattatgttaagttgctcca atccctgtca 840 cttcaggaca tcaagtcaga agattggtct cgctttgttgctccattttg gccagcagtg 900 atacgctcag ccttcacatg gaagggtcta acttcactcttgagcagtgg acttaaaacc 960 ataaaaggag ctttggctat gccattgatg atagagggatacaagaaaga tctaattaag 1020 tttgccatca ttacatgtcg aaaacctgaa taa 1053 131053 DNA Glycine max 13 atggccaccg tggtgaggat cccaacaatc tcatgcatccacatccacac gttccgttcc 60 caatcccctc gcactttcgc cagaatccgg gtcggacccaggtcgtgggc tcctattcgg 120 gcatcggcag cgagctcgga gagaggggag atagtattggagcagaagcc gaagaaggat 180 gacaaggaga aactgcagaa gggaatcgca gagttttacgacgagtcttc tggcttatgg 240 gagaacattt ggggcgacca catgcaccat ggcttttatgacccggattc cactgtttcg 300 ctttcggatc atcgtgctgc tcagatccga atgatccaagagtctcttcg ctttgcctct 360 gtttctgagg agcgtagtaa atggcccaag agtatagttgatgttgggtg tggcataggt 420 ggcagctcca gatacctggc caagaaattt ggagcaaccagtgtaggcat cactctgagt 480 cctgttcaag ctcaaagagc aaatgctctt gctgctgctcaaggattggc tgataaggtt 540 tcctttcagg ttgctgacgc tctacagcaa ccattctctgacggccagtt tgatctggtg 600 tggtccatgg agagtggaga gcatatgcct gacaaagctaagtttgttgg agagttagct 660 cgggtagcag caccaggtgc cactataata atagtaacatggtgccacag ggatcttggc 720 cctgacgaac aatccttaca tccatgggag caagatctcttaaagaagat ttgcgatgca 780 tattacctcc ctgcctggtg ctcaacttct gattatgttaagttgctcca atccctgtca 840 cttcaggaca tcaagtcaga agattggtct cgctttggtgctccattttg gccagcagtg 900 atacgctcag ccttcacatg gaagggtcta acttcactcttgagcagtgg ccaaaaaacg 960 ataaaaggag ctttggctat gccattgatg atagagggatacaagaaaga tctaattaag 1020 tttgccatca ttacatgtcg aaaacctgaa taa 1053 14933 DNA Tagetes erecta 14 gcccttagcg tggtcgcggc cgaggtacca gttacggttactccggcgac gacgaaggcg 60 gaggatgtgg agctgaagaa aggaattgca gagttctacgatgaatcgtc ggagatgtgg 120 gagaatatat ggggagaaca catgcatcat ggatactataacactaatgc cgttgttgaa 180 ctctccgatc atcgttctgc tcagatccgt atgattgaacaagccctact tttcgcatct 240 gtttcagatg atccagtaaa gaaacctaga agcatcgttgatgttgggtg tggcataggt 300 ggtagctcaa ggtatctggc aaagaaatac gaagctgaatgccatggaat cactctcagc 360 cctgtgcaag ctgagagagc tcaagctcta gctgctgctcaaggattggc cgataaggct 420 tcatttcaag ttgctgatgc tttagaccaa ccatttcctgatggaaagtt tgatctggtc 480 tggtcaatgg agagtggtga acacatgcct gacaaactaaagtttgttag tgagttggtt 540 cgggttgctg ccccaggagc cacgattatc atagttacatggtgccatag ggatctttct 600 cctggtgaaa agtcccttcg acccgatgaa gaaaaaatcttgaaaaagat ttgttccagc 660 ttttatcttc ctgcttggtg ttcaacatct gattatgtaaaattactaga gtccctttct 720 cttcaggaca tcaaagctgc agactggtca gcaaacgtggctccattttg gcctgctgta 780 ataaaaacag cattatcttg gaagggcatt acttcgctacttcgtagtgg atggaagtca 840 ataagagggg caatggtaat gccattgatg attgaaggatttaagaagga tataatcaaa 900 ttctccatca tcacatgcaa aaagcctgaa taa 933 151230 DNA Sorghum bicolor 15 cgaacggcga gcagcaggag ggcgtcgcga acccttgggcggcggatcgg tacccgtagg 60 cagccactac tactaccgcg ccccttcgca cgtcccgcgccgctcccgcc cccgcggacg 120 cggcggcgtc gtcagcctgc gtccgatggc ctcgtcgacggcggctcagc cccccgcgcc 180 ggcgcccccg ggcctgaagg agggcatcgc ggggctgtacgacgagtctt cggggctgtg 240 ggagaacatc tggggcgacc acatgcacca cggcttctacgactcgggcg aggccgcgtc 300 catggccgac caccgacgcg cccagatccg catgatcgaggaggcgctcg ccttcgccgc 360 cgtcccatcc ccagatgatc cggagaaggc accaaaaaccatagtagatg ttggatgtgg 420 cattggtggt agctcaaggt acttggctaa gaaatacggagcacagtgca aggggatcac 480 attgagccct gttcaagctg aaagaggaaa tgctcttgctacagcgcagg ggttgtcgga 540 tcaggttact ctgcaagttg ctgatgctct ggagcaaccgtttcctgatg ggcagtttga 600 tctggtatgg tccatggaga gtggcgagca catgccggacaagagaaagt ttgttagtga 660 gctggcacgc gtcgctgctc ctggagggac aataatcatcgtgacatggt gccataggaa 720 cctcgaacca tctgagactt cgctaaaacc cgatgaactgagtctcttga agaggatttg 780 cgatgcgtac tacctcccag actggtgctc accttcagactatgtgaaca tcgccaaatc 840 actgtctctg gaggatatca aggcagctga ttggtcagagaatgtggccc cattttggcc 900 cgctgtgata aaatcagcac taacatggaa gggcctcacctctctactga caagcggatg 960 gaagacgatc agaggggcga tggtgatgcc gctgatgatccaaggttaca agaaggggct 1020 catcaaattc accatcatca cctgtcgcaa gcctggagcagcgtaggtga ccaaggggca 1080 gaagttactg tcaaagcacc tctgctaagt ccaataatgtagatccatgg ccccatcacc 1140 gtctattgta ctgtactgta ctgtaccaga atgaacagtctcctgggaca tgttttccaa 1200 ttgccatgac atgtcaaatg atcttctacc 1230 16 843DNA Nostoc punctiforme 16 atgagtgcaa cactttacca gcaaattcag caattttacgatgcttcatc tggtctgtgg 60 gaacagatat ggggcgaaca catgcaccac ggctattacggcgctgatgg tacccagaaa 120 aaagaccgcc gtcaggctca aattgattta atcgaagaattgcttaattg ggcaggggta 180 caagcagcag aagatatact agatgtgggt tgtggaattggcggtagttc tttatacctg 240 gcgcaaaagt ttaatgctaa agctacaggg attacattgagtcctgtaca agctgcaaga 300 gcaacagaac gcgcattgga agctaatttg agtctgagaacacagttcca agtcgctaat 360 gctcaagcaa tgccctttgc tgacgattct tttgacttggtttggtcgct ggaaagtggc 420 gaacacatgc cagataaaac caagtttctt caggagtgctatcgagtact gaagcctggt 480 ggcaagttaa ttatggtgac ttggtgtcat cgaccaactgatgaatctcc attaacggca 540 gatgaggaaa agcacttgca ggatatttat cgggtgtattgtttgcctta tgtgatttct 600 ttgccagagt atgaagcgat cgcacatcaa ctaccattacataatatccg cactgctgat 660 tggtcaactg ctgtcgcccc cttttggaat gtggtaattgattctgcatt cactccccaa 720 gcgctttggg gtttactaaa tgctggttgg actaccattcaaggggcatt atcactggga 780 ttaatgcgtc gcggttatga acgtgggtta attcggtttggcttactgtg cggcaataag 840 tag 843 17 843 DNA Anabaena sp. 17 atgagtgcaacactttacca acaaattcag caattttacg atgcttcctc tgggctgtgg 60 gaagagatttggggcgaaca tatgcaccac ggctattatg gtgcagacgg tactgaacaa 120 aaaaaccgccgtcaggcgca aattgattta attgaagaat tactcacttg ggcaggagta 180 caaacagcagaaaatatact agatgtgggt tgtggtattg gtggtagttc tctgtatttg 240 gcaggaaagttgaatgctaa agctacagga attaccctga gtccagtgca agccgctaga 300 gccacagaaagagccaagga agctggttta agtggtagaa gtcagttttt agtggcaaat 360 gcccaagcaatgccttttga tgataattct tttgacttgg tgtggtcgct agaaagtggc 420 gaacatatgccagataaaac caagtttttg caagagtgtt atcgagtctt gaaaccgggc 480 ggtaagttaatcatggtgac atggtgtcat cgtcccactg ataaaacacc actgacggct 540 gatgaaaaaaaacacctaga agatatttat cgggtgtatt gtttgcctta tgtaatttcg 600 ttgccggagtatgaagcgat cgcacgtcaa ctaccattaa ataatatccg caccgccgac 660 tggtcgcaatccgtcgccca attttggaac atagtcatcg attccgcctt taccccccaa 720 gcaatattcggcttactccg cgcaggttgg actaccatcc aaggagcctt atcactaggc 780 ttaatgcgtcgcggctatga gcgcgggtta attcggtttg ggttgctttg tggggataag 840 tga 843 18348 PRT Arabidopsis thaliana 18 Met Lys Ala Thr Leu Ala Ala Pro Ser SerLeu Thr Ser Leu Pro Tyr 1 5 10 15 Arg Thr Asn Ser Ser Phe Gly Ser LysSer Ser Leu Leu Phe Arg Ser 20 25 30 Pro Ser Ser Ser Ser Ser Val Ser MetThr Thr Thr Arg Gly Asn Val 35 40 45 Ala Val Ala Ala Ala Ala Thr Ser ThrGlu Ala Leu Arg Lys Gly Ile 50 55 60 Ala Glu Phe Tyr Asn Glu Thr Ser GlyLeu Trp Glu Glu Ile Trp Gly 65 70 75 80 Asp His Met His His Gly Phe TyrAsp Pro Asp Ser Ser Val Gln Leu 85 90 95 Ser Asp Ser Gly His Lys Glu AlaGln Ile Arg Met Ile Glu Glu Ser 100 105 110 Leu Arg Phe Ala Gly Val ThrAsp Glu Glu Glu Glu Lys Lys Ile Lys 115 120 125 Lys Val Val Asp Val GlyCys Gly Ile Gly Gly Ser Ser Arg Tyr Leu 130 135 140 Ala Ser Lys Phe GlyAla Glu Cys Ile Gly Ile Thr Leu Ser Pro Val 145 150 155 160 Gln Ala LysArg Ala Asn Asp Leu Ala Ala Ala Gln Ser Leu Ser His 165 170 175 Lys AlaSer Phe Gln Val Ala Asp Ala Leu Asp Gln Pro Phe Glu Asp 180 185 190 GlyLys Phe Asp Leu Val Trp Ser Met Glu Ser Gly Glu His Met Pro 195 200 205Asp Lys Ala Lys Phe Val Lys Glu Leu Val Arg Val Ala Ala Pro Gly 210 215220 Gly Arg Ile Ile Ile Val Thr Trp Cys His Arg Asn Leu Ser Ala Gly 225230 235 240 Glu Glu Ala Leu Gln Pro Trp Glu Gln Asn Ile Leu Asp Lys IleCys 245 250 255 Lys Thr Phe Tyr Leu Pro Ala Trp Cys Ser Thr Asp Asp TyrVal Asn 260 265 270 Leu Leu Gln Ser His Ser Leu Gln Asp Ile Lys Cys AlaAsp Trp Ser 275 280 285 Glu Asn Val Ala Pro Phe Trp Pro Ala Val Ile ArgThr Ala Leu Thr 290 295 300 Trp Lys Gly Leu Val Ser Leu Leu Arg Ser GlyMet Lys Ser Ile Lys 305 310 315 320 Gly Ala Leu Thr Met Pro Leu Met IleGlu Gly Tyr Lys Lys Gly Val 325 330 335 Ile Lys Phe Gly Ile Ile Thr CysGln Lys Pro Leu 340 345 19 348 PRT Arabidopsis thaliana 19 Met Lys AlaThr Leu Ala Ala Pro Ser Ser Leu Thr Ser Leu Pro Tyr 1 5 10 15 Arg ThrAsn Ser Ser Phe Gly Ser Lys Ser Ser Leu Leu Phe Arg Ser 20 25 30 Pro SerSer Ser Ser Ser Val Ser Met Thr Thr Thr Arg Gly Asn Val 35 40 45 Ala ValAla Ala Ala Ala Thr Ser Thr Glu Ala Leu Arg Lys Gly Ile 50 55 60 Ala GluPhe Tyr Asn Glu Thr Ser Gly Leu Trp Glu Glu Ile Trp Gly 65 70 75 80 AspHis Met His His Gly Phe Tyr Asp Pro Asp Ser Ser Val Gln Leu 85 90 95 SerAsp Ser Gly His Lys Glu Ala Gln Ile Arg Met Ile Glu Glu Ser 100 105 110Leu Arg Phe Ala Gly Val Thr Asp Glu Glu Glu Glu Lys Lys Ile Lys 115 120125 Lys Val Val Asp Val Gly Cys Gly Ile Gly Gly Ser Ser Arg Tyr Leu 130135 140 Ala Ser Lys Phe Gly Ala Glu Cys Ile Gly Ile Thr Leu Ser Pro Val145 150 155 160 Gln Ala Lys Arg Ala Asn Asp Leu Ala Ala Ala Gln Ser LeuAla His 165 170 175 Lys Ala Ser Phe Gln Val Ala Asp Ala Leu Asp Gln ProPhe Glu Asp 180 185 190 Gly Lys Phe Asp Leu Val Trp Ser Met Glu Ser GlyGlu His Met Pro 195 200 205 Asp Lys Ala Lys Phe Val Lys Glu Leu Val ArgVal Ala Ala Pro Gly 210 215 220 Gly Arg Ile Ile Ile Val Thr Trp Cys HisArg Asn Leu Ser Ala Gly 225 230 235 240 Glu Glu Ala Leu Gln Pro Trp GluGln Asn Ile Leu Asp Lys Ile Cys 245 250 255 Lys Thr Phe Tyr Leu Pro AlaTrp Cys Ser Thr Asp Asp Tyr Val Asn 260 265 270 Leu Leu Gln Ser His SerLeu Gln Asp Ile Lys Cys Ala Asp Trp Ser 275 280 285 Glu Asn Val Ala ProPhe Trp Pro Ala Val Ile Arg Thr Ala Leu Thr 290 295 300 Trp Lys Gly LeuVal Ser Leu Leu Arg Ser Gly Met Lys Ser Ile Lys 305 310 315 320 Gly AlaLeu Thr Met Pro Leu Met Ile Glu Gly Tyr Lys Lys Gly Val 325 330 335 IleLys Phe Gly Ile Ile Thr Cys Gln Lys Pro Leu 340 345 20 364 PRT Oryzasativa 20 Met Ala His Ala Ala Ala Ala Thr Gly Ala Leu Ala Pro Leu HisPro 1 5 10 15 Leu Leu Arg Cys Thr Ser Arg His Leu Cys Ala Ser Ala SerPro Arg 20 25 30 Ala Gly Leu Cys Leu His His His Arg Arg Arg Arg Arg SerSer Arg 35 40 45 Arg Thr Lys Leu Ala Val Arg Ala Met Ala Pro Thr Leu SerSer Ser 50 55 60 Ser Thr Ala Ala Ala Ala Pro Pro Gly Leu Lys Glu Gly IleAla Gly 65 70 75 80 Leu Tyr Asp Glu Ser Ser Gly Val Trp Glu Ser Ile TrpGly Glu His 85 90 95 Met His His Gly Phe Tyr Asp Ala Gly Glu Ala Ala SerMet Ser Asp 100 105 110 His Arg Arg Ala Gln Ile Arg Met Ile Glu Glu SerLeu Ala Phe Ala 115 120 125 Ala Val Pro Gly Ala Asp Asp Ala Glu Lys LysPro Lys Ser Val Val 130 135 140 Asp Val Gly Cys Gly Ile Gly Gly Ser SerArg Tyr Leu Ala Asn Lys 145 150 155 160 Tyr Gly Ala Gln Cys Tyr Gly IleThr Leu Ser Pro Val Gln Ala Glu 165 170 175 Arg Gly Asn Ala Leu Ala AlaGlu Gln Gly Leu Ser Asp Lys Val Arg 180 185 190 Ile Gln Val Gly Asp AlaLeu Glu Gln Pro Phe Pro Asp Gly Gln Phe 195 200 205 Asp Leu Val Trp SerMet Glu Ser Gly Glu His Met Pro Asp Lys Arg 210 215 220 Gln Phe Val SerGlu Leu Ala Arg Val Ala Ala Pro Gly Ala Arg Ile 225 230 235 240 Ile IleVal Thr Trp Cys His Arg Asn Leu Glu Pro Ser Glu Glu Ser 245 250 255 LeuLys Pro Asp Glu Leu Asn Leu Leu Lys Arg Ile Cys Asp Ala Tyr 260 265 270Tyr Leu Pro Asp Trp Cys Ser Pro Ser Asp Tyr Val Lys Ile Ala Glu 275 280285 Ser Leu Ser Leu Glu Asp Ile Arg Thr Ala Asp Trp Ser Glu Asn Val 290295 300 Ala Pro Phe Trp Pro Ala Val Ile Lys Ser Ala Leu Thr Trp Lys Gly305 310 315 320 Leu Thr Ser Leu Leu Arg Ser Gly Trp Lys Thr Ile Arg GlyAla Met 325 330 335 Val Met Pro Leu Met Ile Glu Gly Tyr Lys Lys Gly LeuIle Lys Phe 340 345 350 Thr Ile Ile Thr Cys Arg Lys Pro Glu Thr Thr Gln355 360 21 352 PRT Zea mays 21 Met Ala His Ala Ala Leu Leu His Cys SerGln Ser Ser Arg Ser Leu 1 5 10 15 Ala Ala Cys Arg Arg Gly Ser His TyrArg Ala Pro Ser His Val Pro 20 25 30 Arg His Ser Arg Arg Leu Arg Arg AlaVal Val Ser Leu Arg Pro Met 35 40 45 Ala Ser Ser Thr Ala Gln Ala Pro AlaThr Ala Pro Pro Gly Leu Lys 50 55 60 Glu Gly Ile Ala Gly Leu Tyr Asp GluSer Ser Gly Leu Trp Glu Asn 65 70 75 80 Ile Trp Gly Asp His Met His HisGly Phe Tyr Asp Ser Ser Glu Ala 85 90 95 Ala Ser Met Ala Asp His Arg ArgAla Gln Ile Arg Met Ile Glu Glu 100 105 110 Ala Leu Ala Phe Ala Gly ValPro Ala Ser Asp Asp Pro Glu Lys Thr 115 120 125 Pro Lys Thr Ile Val AspVal Gly Cys Gly Ile Gly Gly Ser Ser Arg 130 135 140 Tyr Leu Ala Lys LysTyr Gly Ala Gln Cys Thr Gly Ile Thr Leu Ser 145 150 155 160 Pro Val GlnAla Glu Arg Gly Asn Ala Leu Ala Ala Ala Gln Gly Leu 165 170 175 Ser AspGln Val Thr Leu Gln Val Ala Asp Ala Leu Glu Gln Pro Phe 180 185 190 ProAsp Gly Gln Phe Asp Leu Val Trp Ser Met Glu Ser Gly Glu His 195 200 205Met Pro Asp Lys Arg Lys Phe Val Ser Glu Leu Ala Arg Val Ala Ala 210 215220 Pro Gly Gly Thr Ile Ile Ile Val Thr Trp Cys His Arg Asn Leu Asp 225230 235 240 Pro Ser Glu Thr Ser Leu Lys Pro Asp Glu Leu Ser Leu Leu ArgArg 245 250 255 Ile Cys Asp Ala Tyr Tyr Leu Pro Asp Trp Cys Ser Pro SerAsp Tyr 260 265 270 Val Asn Ile Ala Lys Ser Leu Ser Leu Glu Asp Ile LysThr Ala Asp 275 280 285 Trp Ser Glu Asn Val Ala Pro Phe Trp Pro Ala ValIle Lys Ser Ala 290 295 300 Leu Thr Trp Lys Gly Phe Thr Ser Leu Leu ThrThr Gly Trp Lys Thr 305 310 315 320 Ile Arg Gly Ala Met Val Met Pro LeuMet Ile Gln Gly Tyr Lys Lys 325 330 335 Gly Leu Ile Lys Phe Thr Ile IleThr Cys Arg Lys Pro Gly Ala Ala 340 345 350 22 345 PRT Gossypiumhirsutum 22 Met Ala Ala Ala Leu Gln Leu Gln Thr His Pro Cys Phe His GlyThr 1 5 10 15 Cys Gln Leu Ser Pro Pro Pro Arg Pro Ser Val Ser Phe ProSer Ser 20 25 30 Ser Arg Ser Phe Pro Ser Ser Arg Arg Ser Leu Ser Ala HisVal Lys 35 40 45 Ala Ala Ala Ser Ser Leu Ser Thr Thr Thr Leu Gln Glu GlyIle Ala 50 55 60 Glu Phe Tyr Asp Glu Ser Ser Gly Ile Trp Glu Asp Ile TrpGly Asp 65 70 75 80 His Met His His Gly Tyr Tyr Glu Pro Gly Ser Asp IleSer Gly Ser 85 90 95 Asp His Arg Ala Ala Gln Ile Arg Met Val Glu Glu SerLeu Arg Phe 100 105 110 Ala Gly Ile Ser Glu Asp Pro Ala Asn Arg Pro LysArg Ile Val Asp 115 120 125 Val Gly Cys Gly Ile Gly Gly Ser Ser Arg TyrLeu Ala Arg Lys Tyr 130 135 140 Gly Ala Lys Cys Gln Gly Ile Thr Leu SerPro Val Gln Ala Gly Arg 145 150 155 160 Ala Asn Ala Leu Ala Asn Ala GlnGly Leu Ala Glu Gln Val Cys Phe 165 170 175 Glu Val Ala Asp Ala Leu AsnGln Pro Phe Pro Asp Asp Gln Phe Asp 180 185 190 Leu Val Trp Ser Met GluSer Gly Glu His Met Pro Asp Lys Pro Lys 195 200 205 Phe Val Lys Glu LeuVal Arg Val Ala Ala Pro Gly Gly Thr Ile Ile 210 215 220 Val Val Thr TrpCys His Arg Asp Leu Gly Pro Ser Glu Glu Ser Leu 225 230 235 240 Gln ProTrp Glu Gln Lys Leu Leu Asn Arg Ile Cys Asp Ala Tyr Tyr 245 250 255 LeuPro Glu Trp Cys Ser Thr Ser Asp Tyr Val Lys Leu Phe Gln Ser 260 265 270Leu Ser Leu Gln Asp Ile Lys Ala Gly Asp Trp Thr Glu Asn Val Ala 275 280285 Pro Phe Trp Pro Ala Val Ile Arg Ser Ala Leu Thr Trp Lys Gly Phe 290295 300 Thr Ser Leu Leu Arg Ser Gly Leu Lys Thr Ile Lys Gly Ala Leu Val305 310 315 320 Met Pro Leu Met Ile Glu Gly Phe Gln Lys Gly Val Ile LysPhe Ala 325 330 335 Ile Ile Ala Cys Arg Lys Pro Ala Glu 340 345 23 376PRT Cuphea pulcherrima 23 Met Pro Ile Thr Ser Ile Ser Ala Asn Gln ArgPro Phe Phe Pro Ser 1 5 10 15 Pro Tyr Arg Gly Ser Ser Lys Asn Met AlaPro Pro Glu Leu Ala Gln 20 25 30 Ser Gln Val Pro Met Gly Ser Asn Lys SerAsn Lys Asn His Gly Leu 35 40 45 Val Gly Ser Val Ser Gly Trp Arg Arg MetPhe Gly Thr Trp Ala Thr 50 55 60 Ala Asp Lys Thr Gln Ser Thr Asp Thr SerAsn Glu Gly Val Val Ser 65 70 75 80 Tyr Asp Thr Gln Val Leu Gln Lys GlyIle Ala Glu Phe Tyr Asp Glu 85 90 95 Ser Ser Gly Ile Trp Glu Asp Ile TrpGly Asp His Met His His Gly 100 105 110 Tyr Tyr Asp Gly Ser Thr Pro ValSer Leu Pro Asp His Arg Ser Ala 115 120 125 Gln Ile Arg Met Ile Asp GluAla Leu Arg Phe Ala Ser Val Pro Ser 130 135 140 Gly Glu Glu Asp Glu SerLys Ser Lys Ile Pro Lys Arg Ile Val Asp 145 150 155 160 Val Gly Cys GlyIle Gly Gly Ser Ser Arg Tyr Leu Ala Arg Lys Tyr 165 170 175 Gly Ala GluCys Arg Gly Ile Thr Leu Ser Pro Val Gln Ala Glu Arg 180 185 190 Gly AsnSer Leu Ala Arg Ser Gln Gly Leu Ser Asp Lys Val Ser Phe 195 200 205 GlnVal Ala Asp Ala Leu Ala Gln Pro Phe Pro Asp Gly Gln Phe Asp 210 215 220Leu Val Trp Ser Met Glu Ser Gly Glu His Met Pro Asp Lys Ser Lys 225 230235 240 Phe Val Asn Glu Leu Val Arg Val Ala Ala Pro Gly Gly Thr Ile Ile245 250 255 Ile Val Thr Trp Cys His Arg Asp Leu Arg Glu Asp Glu Asp AlaLeu 260 265 270 Gln Pro Arg Glu Lys Glu Ile Leu Asp Lys Ile Cys Asn ProPhe Tyr 275 280 285 Leu Pro Ala Trp Cys Ser Ala Ala Asp Tyr Val Lys LeuLeu Gln Ser 290 295 300 Leu Asp Val Glu Asp Ile Lys Ser Ala Asp Trp ThrPro Tyr Val Ala 305 310 315 320 Pro Phe Trp Pro Ala Val Leu Lys Ser AlaPhe Thr Ile Lys Gly Phe 325 330 335 Val Ser Leu Leu Arg Ser Gly Met LysThr Ile Lys Gly Ala Phe Ala 340 345 350 Met Pro Leu Met Ile Glu Gly TyrLys Lys Gly Val Ile Lys Phe Ser 355 360 365 Ile Ile Thr Cys Arg Lys ProGlu 370 375 24 347 PRT Brassica napus 24 Met Lys Ala Thr Leu Ala Pro SerSer Leu Ile Ser Leu Pro Arg His 1 5 10 15 Lys Val Ser Ser Leu Arg SerPro Ser Leu Leu Leu Gln Ser Gln Arg 20 25 30 Pro Ser Ser Ala Leu Met ThrThr Thr Thr Ala Ser Arg Gly Ser Val 35 40 45 Ala Val Thr Ala Ala Ala ThrSer Ser Val Glu Ala Leu Arg Glu Gly 50 55 60 Ile Ala Glu Phe Tyr Asn GluThr Ser Gly Leu Trp Glu Glu Ile Trp 65 70 75 80 Gly Asp His Met His HisGly Phe Tyr Asp Pro Asp Ser Ser Val Gln 85 90 95 Leu Ser Asp Ser Gly HisArg Glu Ala Gln Ile Arg Met Ile Glu Glu 100 105 110 Ser Leu Arg Phe AlaGly Val Thr Glu Glu Glu Lys Lys Ile Lys Arg 115 120 125 Val Val Asp ValGly Cys Gly Ile Gly Gly Ser Ser Arg Tyr Ile Ala 130 135 140 Ser Lys PheGly Ala Glu Cys Ile Gly Ile Thr Leu Ser Pro Val Gln 145 150 155 160 AlaLys Arg Ala Asn Asp Leu Ala Ala Ala Gln Ser Leu Ser His Lys 165 170 175Val Ser Phe Gln Val Ala Asp Ala Leu Glu Gln Pro Phe Glu Asp Gly 180 185190 Ile Phe Asp Leu Val Trp Ser Met Glu Ser Gly Glu His Met Pro Asp 195200 205 Lys Ala Lys Phe Val Lys Glu Leu Val Arg Val Ala Ala Pro Gly Gly210 215 220 Arg Ile Ile Ile Val Thr Trp Cys His Arg Asn Leu Ser Pro GlyGlu 225 230 235 240 Glu Ala Leu Gln Pro Trp Glu Gln Asn Leu Leu Asp ArgIle Cys Lys 245 250 255 Thr Phe Tyr Leu Pro Ala Trp Cys Ser Thr Ser AspTyr Val Asp Leu 260 265 270 Leu Gln Ser Leu Ser Leu Gln Asp Ile Lys CysAla Asp Trp Ser Glu 275 280 285 Asn Val Ala Pro Phe Trp Pro Ala Val IleArg Thr Ala Leu Thr Trp 290 295 300 Lys Gly Leu Val Ser Leu Leu Arg SerGly Met Lys Ser Ile Lys Gly 305 310 315 320 Ala Leu Thr Met Pro Leu MetIle Glu Gly Tyr Lys Lys Gly Val Ile 325 330 335 Lys Phe Gly Ile Ile ThrCys Gln Lys Pro Leu 340 345 25 347 PRT Brassica napus 25 Met Lys Ala ThrLeu Ala Pro Pro Ser Ser Leu Ile Ser Leu Pro Arg 1 5 10 15 His Lys ValSer Ser Leu Arg Ser Pro Ser Leu Leu Leu Gln Ser Gln 20 25 30 Arg Arg SerSer Ala Leu Met Thr Thr Thr Ala Ser Arg Gly Ser Val 35 40 45 Ala Val ThrAla Ala Ala Thr Ser Ser Ala Glu Ala Leu Arg Glu Gly 50 55 60 Ile Ala GluPhe Tyr Asn Glu Thr Ser Gly Leu Trp Glu Glu Ile Trp 65 70 75 80 Gly AspHis Met His His Gly Phe Tyr Asp Pro Asp Ser Ser Val Gln 85 90 95 Leu SerAsp Ser Gly His Arg Glu Ala Gln Ile Arg Met Ile Glu Glu 100 105 110 SerLeu Arg Phe Ala Gly Val Thr Glu Glu Glu Lys Lys Ile Lys Arg 115 120 125Val Val Asp Val Gly Cys Gly Ile Gly Gly Ser Ser Arg Tyr Ile Ala 130 135140 Ser Lys Phe Gly Ala Glu Cys Ile Gly Ile Thr Leu Ser Pro Val Gln 145150 155 160 Ala Lys Arg Ala Asn Asp Leu Ala Thr Ala Gln Ser Leu Ser HisLys 165 170 175 Val Ser Phe Gln Val Ala Asp Ala Leu Asp Gln Pro Phe GluAsp Gly 180 185 190 Ile Ser Asp Leu Val Trp Ser Met Glu Ser Gly Glu HisMet Pro Asp 195 200 205 Lys Ala Lys Phe Val Lys Glu Leu Val Arg Val ThrAla Pro Gly Gly 210 215 220 Arg Ile Ile Ile Val Thr Trp Cys His Arg AsnLeu Ser Gln Gly Glu 225 230 235 240 Glu Ser Leu Gln Pro Trp Glu Gln AsnLeu Leu Asp Arg Ile Cys Lys 245 250 255 Thr Phe Tyr Leu Pro Ala Trp CysSer Thr Thr Asp Tyr Val Glu Leu 260 265 270 Leu Gln Ser Leu Ser Leu GlnAsp Ile Lys Tyr Ala Asp Trp Ser Glu 275 280 285 Asn Val Ala Pro Phe TrpPro Ala Val Ile Arg Thr Ala Leu Thr Trp 290 295 300 Lys Gly Leu Val SerLeu Leu Arg Ser Gly Met Lys Ser Ile Lys Gly 305 310 315 320 Ala Leu ThrMet Pro Leu Met Ile Glu Gly Tyr Lys Lys Gly Val Ile 325 330 335 Lys PheGly Ile Ile Thr Cys Gln Lys Pro Leu 340 345 26 310 PRT Lycopersiconesculentum 26 Met Ala Ser Val Ala Ala Met Asn Ala Val Ser Ser Ser SerVal Glu 1 5 10 15 Val Gly Ile Gln Asn Gln Gln Glu Leu Lys Lys Gly IleAla Asp Leu 20 25 30 Tyr Asp Glu Ser Ser Gly Ile Trp Glu Asp Ile Trp GlyAsp His Met 35 40 45 His His Gly Tyr Tyr Glu Pro Lys Ser Ser Val Glu LeuSer Asp His 50 55 60 Arg Ala Ala Gln Ile Arg Met Ile Glu Gln Ala Leu SerPhe Ala Ala 65 70 75 80 Ile Ser Glu Asp Pro Ala Lys Lys Pro Thr Ser IleVal Asp Val Gly 85 90 95 Cys Gly Ile Gly Gly Ser Ser Arg Tyr Leu Ala LysLys Tyr Gly Ala 100 105 110 Thr Ala Lys Gly Ile Thr Leu Ser Pro Val GlnAla Glu Arg Ala Gln 115 120 125 Ala Leu Ala Asp Ala Gln Gly Leu Gly AspLys Val Ser Phe Gln Val 130 135 140 Ala Asp Ala Leu Asn Gln Pro Phe ProAsp Gly Gln Phe Asp Leu Val 145 150 155 160 Trp Ser Met Glu Ser Gly GluHis Met Pro Asn Lys Glu Lys Phe Val 165 170 175 Gly Glu Leu Ala Arg ValAla Ala Pro Gly Gly Thr Ile Ile Leu Val 180 185 190 Thr Trp Cys His ArgAsp Leu Ser Pro Ser Glu Glu Ser Leu Thr Pro 195 200 205 Glu Glu Lys GluLeu Leu Asn Lys Ile Cys Lys Ala Phe Tyr Leu Pro 210 215 220 Ala Trp CysSer Thr Ala Asp Tyr Val Lys Leu Leu Gln Ser Asn Ser 225 230 235 240 LeuGln Asp Ile Lys Ala Glu Asp Trp Ser Glu Asn Val Ala Pro Phe 245 250 255Trp Pro Ala Val Ile Lys Ser Ala Leu Thr Trp Lys Gly Phe Thr Ser 260 265270 Val Leu Arg Ser Gly Trp Lys Thr Ile Lys Ala Ala Leu Ala Met Pro 275280 285 Leu Met Ile Glu Gly Tyr Lys Lys Gly Leu Ile Lys Phe Ala Ile Ile290 295 300 Thr Cys Arg Lys Pro Glu 305 310 27 302 PRT Glycine max 27Met Ser Val Glu Gln Lys Ala Ala Gly Lys Glu Glu Glu Gly Lys Leu 1 5 1015 Gln Lys Gly Ile Ala Glu Phe Tyr Asp Glu Ser Ser Gly Ile Trp Glu 20 2530 Asn Ile Trp Gly Asp His Met His His Gly Phe Tyr Asp Pro Asp Ser 35 4045 Thr Val Ser Val Ser Asp His Arg Ala Ala Gln Ile Arg Met Ile Gln 50 5560 Glu Ser Leu Arg Phe Ala Ser Leu Leu Ser Glu Asn Pro Ser Lys Trp 65 7075 80 Pro Lys Ser Ile Val Asp Val Gly Cys Gly Ile Gly Gly Ser Ser Arg 8590 95 Tyr Leu Ala Lys Lys Phe Gly Ala Thr Ser Val Gly Ile Thr Leu Ser100 105 110 Pro Val Gln Ala Gln Arg Ala Asn Ala Leu Ala Ala Ala Gln GlyLeu 115 120 125 Ala Asp Lys Val Ser Phe Gln Val Ala Asp Ala Leu Gln GlnPro Phe 130 135 140 Ser Asp Gly Gln Phe Asp Leu Val Trp Ser Met Glu SerGly Glu His 145 150 155 160 Met Pro Asp Lys Ala Lys Phe Val Gly Glu LeuAla Arg Val Ala Ala 165 170 175 Pro Gly Ala Thr Ile Ile Ile Val Thr TrpCys His Arg Asp Leu Gly 180 185 190 Pro Asp Glu Gln Ser Leu His Pro TrpGlu Gln Asp Leu Leu Lys Lys 195 200 205 Ile Cys Asp Ala Tyr Tyr Leu ProAla Trp Cys Ser Thr Ser Asp Tyr 210 215 220 Val Lys Leu Leu Gln Ser LeuSer Leu Gln Asp Ile Lys Ser Glu Asp 225 230 235 240 Trp Ser Arg Phe ValAla Pro Phe Trp Pro Ala Val Ile Arg Ser Ala 245 250 255 Phe Thr Trp LysGly Leu Thr Ser Leu Leu Ser Ser Gly Gln Lys Thr 260 265 270 Ile Lys GlyAla Leu Ala Met Pro Leu Met Ile Glu Gly Tyr Lys Lys 275 280 285 Asp LeuIle Lys Phe Ala Ile Ile Thr Cys Arg Lys Pro Glu 290 295 300 28 350 PRTGlycine max 28 Met Ala Thr Val Val Arg Ile Pro Thr Ile Ser Cys Ile HisIle His 1 5 10 15 Thr Phe Arg Ser Gln Ser Pro Arg Thr Phe Ala Arg IleArg Val Gly 20 25 30 Pro Arg Ser Trp Ala Pro Ile Arg Ala Ser Ala Ala SerSer Glu Arg 35 40 45 Gly Glu Ile Val Leu Glu Gln Lys Pro Lys Lys Glu GluGlu Gly Lys 50 55 60 Leu Gln Lys Gly Ile Ala Glu Phe Tyr Asp Glu Ser SerGly Leu Trp 65 70 75 80 Glu Asn Ile Trp Gly Asp His Met His His Gly PheTyr Asp Pro Asp 85 90 95 Ser Thr Val Ser Val Ser Asp His Arg Ala Ala GlnIle Arg Met Ile 100 105 110 Gln Glu Ser Leu Arg Phe Ala Ser Val Ser GluGlu Arg Ser Lys Trp 115 120 125 Pro Lys Ser Ile Val Asp Val Gly Cys GlyIle Gly Gly Ser Ser Arg 130 135 140 Tyr Leu Ala Lys Lys Phe Gly Ala ThrSer Val Gly Ile Thr Leu Ser 145 150 155 160 Pro Val Gln Ala Gln Arg AlaAsn Ala Leu Ala Ala Ala Gln Gly Leu 165 170 175 Ala Asp Lys Val Ser PheGln Val Ala Asp Ala Leu Gln Gln Pro Phe 180 185 190 Ser Asp Gly Gln PheAsp Leu Val Trp Ser Met Glu Ser Gly Glu His 195 200 205 Met Pro Asp LysAla Lys Phe Val Gly Glu Leu Ala Arg Val Ala Ala 210 215 220 Pro Gly AlaThr Ile Ile Ile Val Thr Trp Cys His Arg Asp Leu Gly 225 230 235 240 ProAsp Glu Gln Ser Leu His Pro Trp Glu Gln Asp Leu Leu Lys Lys 245 250 255Ile Cys Asp Ala Tyr Tyr Leu Pro Ala Trp Cys Ser Thr Ser Asp Tyr 260 265270 Val Lys Leu Leu Gln Ser Leu Ser Leu Gln Asp Ile Lys Ser Glu Asp 275280 285 Trp Ser Arg Phe Val Ala Pro Phe Trp Pro Ala Val Ile Arg Ser Ala290 295 300 Phe Thr Trp Lys Gly Leu Thr Ser Leu Leu Ser Ser Gly Leu LysThr 305 310 315 320 Ile Lys Gly Ala Leu Ala Met Pro Leu Met Ile Glu GlyTyr Lys Lys 325 330 335 Asp Leu Ile Lys Phe Ala Ile Ile Thr Cys Arg LysPro Glu 340 345 350 29 350 PRT Glycine max 29 Met Ala Thr Val Val ArgIle Pro Thr Ile Ser Cys Ile His Ile His 1 5 10 15 Thr Phe Arg Ser GlnSer Pro Arg Thr Phe Ala Arg Ile Arg Val Gly 20 25 30 Pro Arg Ser Trp AlaPro Ile Arg Ala Ser Ala Ala Ser Ser Glu Arg 35 40 45 Gly Glu Ile Val LeuGlu Gln Lys Pro Lys Lys Asp Asp Lys Glu Lys 50 55 60 Leu Gln Lys Gly IleAla Glu Phe Tyr Asp Glu Ser Ser Gly Leu Trp 65 70 75 80 Glu Asn Ile TrpGly Asp His Met His His Gly Phe Tyr Asp Pro Asp 85 90 95 Ser Thr Val SerLeu Ser Asp His Arg Ala Ala Gln Ile Arg Met Ile 100 105 110 Gln Glu SerLeu Arg Phe Ala Ser Val Ser Glu Glu Arg Ser Lys Trp 115 120 125 Pro LysSer Ile Val Asp Val Gly Cys Gly Ile Gly Gly Ser Ser Arg 130 135 140 TyrLeu Ala Lys Lys Phe Gly Ala Thr Ser Val Gly Ile Thr Leu Ser 145 150 155160 Pro Val Gln Ala Gln Arg Ala Asn Ala Leu Ala Ala Ala Gln Gly Leu 165170 175 Ala Asp Lys Val Ser Phe Gln Val Ala Asp Ala Leu Gln Gln Pro Phe180 185 190 Ser Asp Gly Gln Phe Asp Leu Val Trp Ser Met Glu Ser Gly GluHis 195 200 205 Met Pro Asp Lys Ala Lys Phe Val Gly Glu Leu Ala Arg ValAla Ala 210 215 220 Pro Gly Ala Thr Ile Ile Ile Val Thr Trp Cys His ArgAsp Leu Gly 225 230 235 240 Pro Asp Glu Gln Ser Leu His Pro Trp Glu GlnAsp Leu Leu Lys Lys 245 250 255 Ile Cys Asp Ala Tyr Tyr Leu Pro Ala TrpCys Ser Thr Ser Asp Tyr 260 265 270 Val Lys Leu Leu Gln Ser Leu Ser LeuGln Asp Ile Lys Ser Glu Asp 275 280 285 Trp Ser Arg Phe Gly Ala Pro PheTrp Pro Ala Val Ile Arg Ser Ala 290 295 300 Phe Thr Trp Lys Gly Leu ThrSer Leu Leu Ser Ser Gly Gln Lys Thr 305 310 315 320 Ile Lys Gly Ala LeuAla Met Pro Leu Met Ile Glu Gly Tyr Lys Lys 325 330 335 Asp Leu Ile LysPhe Ala Ile Ile Thr Cys Arg Lys Pro Glu 340 345 350 30 310 PRT Tageteserecta 30 Ala Leu Ser Val Val Ala Ala Glu Val Pro Val Thr Val Thr ProAla 1 5 10 15 Thr Thr Lys Ala Glu Asp Val Glu Leu Lys Lys Gly Ile AlaGlu Phe 20 25 30 Tyr Asp Glu Ser Ser Glu Met Trp Glu Asn Ile Trp Gly GluHis Met 35 40 45 His His Gly Tyr Tyr Asn Thr Asn Ala Val Val Glu Leu SerAsp His 50 55 60 Arg Ser Ala Gln Ile Arg Met Ile Glu Gln Ala Leu Leu PheAla Ser 65 70 75 80 Val Ser Asp Asp Pro Val Lys Lys Pro Arg Ser Ile ValAsp Val Gly 85 90 95 Cys Gly Ile Gly Gly Ser Ser Arg Tyr Leu Ala Lys LysTyr Glu Ala 100 105 110 Glu Cys His Gly Ile Thr Leu Ser Pro Val Gln AlaGlu Arg Ala Gln 115 120 125 Ala Leu Ala Ala Ala Gln Gly Leu Ala Asp LysAla Ser Phe Gln Val 130 135 140 Ala Asp Ala Leu Asp Gln Pro Phe Pro AspGly Lys Phe Asp Leu Val 145 150 155 160 Trp Ser Met Glu Ser Gly Glu HisMet Pro Asp Lys Leu Lys Phe Val 165 170 175 Ser Glu Leu Val Arg Val AlaAla Pro Gly Ala Thr Ile Ile Ile Val 180 185 190 Thr Trp Cys His Arg AspLeu Ser Pro Gly Glu Lys Ser Leu Arg Pro 195 200 205 Asp Glu Glu Lys IleLeu Lys Lys Ile Cys Ser Ser Phe Tyr Leu Pro 210 215 220 Ala Trp Cys SerThr Ser Asp Tyr Val Lys Leu Leu Glu Ser Leu Ser 225 230 235 240 Leu GlnAsp Ile Lys Ala Ala Asp Trp Ser Ala Asn Val Ala Pro Phe 245 250 255 TrpPro Ala Val Ile Lys Thr Ala Leu Ser Trp Lys Gly Ile Thr Ser 260 265 270Leu Leu Arg Ser Gly Trp Lys Ser Ile Arg Gly Ala Met Val Met Pro 275 280285 Leu Met Ile Glu Gly Phe Lys Lys Asp Ile Ile Lys Phe Ser Ile Ile 290295 300 Thr Cys Lys Lys Pro Glu 305 310 31 354 PRT Sorghum bicolor 31Glu Arg Arg Ala Ala Gly Gly Arg Arg Glu Pro Leu Gly Gly Gly Ser 1 5 1015 Val Pro Val Gly Ser His Tyr Tyr Tyr Arg Ala Pro Ser His Val Pro 20 2530 Arg Arg Ser Arg Pro Arg Gly Arg Gly Gly Val Val Ser Leu Arg Pro 35 4045 Met Ala Ser Ser Thr Ala Ala Gln Pro Pro Ala Pro Ala Pro Pro Gly 50 5560 Leu Lys Glu Gly Ile Ala Gly Leu Tyr Asp Glu Ser Ser Gly Leu Trp 65 7075 80 Glu Asn Ile Trp Gly Asp His Met His His Gly Phe Tyr Asp Ser Gly 8590 95 Glu Ala Ala Ser Met Ala Asp His Arg Arg Ala Gln Ile Arg Met Ile100 105 110 Glu Glu Ala Leu Ala Phe Ala Ala Val Pro Ser Pro Asp Asp ProGlu 115 120 125 Lys Ala Pro Lys Thr Ile Val Asp Val Gly Cys Gly Ile GlyGly Ser 130 135 140 Ser Arg Tyr Leu Ala Lys Lys Tyr Gly Ala Gln Cys LysGly Ile Thr 145 150 155 160 Leu Ser Pro Val Gln Ala Glu Arg Gly Asn AlaLeu Ala Thr Ala Gln 165 170 175 Gly Leu Ser Asp Gln Val Thr Leu Gln ValAla Asp Ala Leu Glu Gln 180 185 190 Pro Phe Pro Asp Gly Gln Phe Asp LeuVal Trp Ser Met Glu Ser Gly 195 200 205 Glu His Met Pro Asp Lys Arg LysPhe Val Ser Glu Leu Ala Arg Val 210 215 220 Ala Ala Pro Gly Gly Thr IleIle Ile Val Thr Trp Cys His Arg Asn 225 230 235 240 Leu Glu Pro Ser GluThr Ser Leu Lys Pro Asp Glu Leu Ser Leu Leu 245 250 255 Lys Arg Ile CysAsp Ala Tyr Tyr Leu Pro Asp Trp Cys Ser Pro Ser 260 265 270 Asp Tyr ValAsn Ile Ala Lys Ser Leu Ser Leu Glu Asp Ile Lys Ala 275 280 285 Ala AspTrp Ser Glu Asn Val Ala Pro Phe Trp Pro Ala Val Ile Lys 290 295 300 SerAla Leu Thr Trp Lys Gly Leu Thr Ser Leu Leu Thr Ser Gly Trp 305 310 315320 Lys Thr Ile Arg Gly Ala Met Val Met Pro Leu Met Ile Gln Gly Tyr 325330 335 Lys Lys Gly Leu Ile Lys Phe Thr Ile Ile Thr Cys Arg Lys Pro Gly340 345 350 Ala Ala 32 92 PRT pea 32 Met Ala Ser Ser Met Leu Ser Ser AlaThr Met Val Ala Ser Pro Ala 1 5 10 15 Gln Ala Thr Met Val Ala Pro PheAsn Gly Leu Lys Ser Ser Ala Ala 20 25 30 Phe Pro Ala Thr Arg Lys Ala AsnAsn Asp Ile Thr Ser Ile Thr Ser 35 40 45 Asn Gly Gly Arg Val Asn Cys MetGln Val Trp Pro Pro Ile Gly Lys 50 55 60 Lys Lys Phe Glu Thr Leu Ser TyrLeu Pro Asp Leu Thr Asp Ser Gly 65 70 75 80 Gly Arg Val Asn Cys Met GlnAla Asn Asn Asn Asn 85 90 33 301 PRT Brassica napus 33 Met Val Ala ValThr Ala Ala Ala Thr Ser Ser Val Glu Ala Leu Arg 1 5 10 15 Glu Gly IleAla Glu Phe Tyr Asn Glu Thr Ser Gly Leu Trp Glu Glu 20 25 30 Ile Trp GlyAsp His Met His His Gly Phe Tyr Asp Pro Asp Ser Ser 35 40 45 Val Gln LeuSer Asp Ser Gly His Arg Glu Ala Gln Ile Arg Met Ile 50 55 60 Glu Glu SerLeu Arg Phe Ala Gly Val Thr Glu Glu Glu Lys Lys Ile 65 70 75 80 Lys ArgVal Val Asp Val Gly Cys Gly Ile Gly Gly Ser Ser Arg Tyr 85 90 95 Ile AlaSer Lys Phe Gly Ala Glu Cys Ile Gly Ile Thr Leu Ser Pro 100 105 110 ValGln Ala Lys Arg Ala Asn Asp Leu Ala Ala Ala Gln Ser Leu Ser 115 120 125His Lys Val Ser Phe Gln Val Ala Asp Ala Leu Glu Gln Pro Phe Glu 130 135140 Asp Gly Ile Phe Asp Leu Val Trp Ser Met Glu Ser Gly Glu His Met 145150 155 160 Pro Asp Lys Ala Lys Phe Val Lys Glu Leu Val Arg Val Ala AlaPro 165 170 175 Gly Gly Arg Ile Ile Ile Val Thr Trp Cys His Arg Asn LeuSer Pro 180 185 190 Gly Glu Glu Ala Leu Gln Pro Trp Glu Gln Asn Leu LeuAsp Arg Ile 195 200 205 Cys Lys Thr Phe Tyr Leu Pro Ala Trp Cys Ser ThrSer Asp Tyr Val 210 215 220 Asp Leu Leu Gln Ser Leu Ser Leu Gln Asp IleLys Cys Ala Asp Trp 225 230 235 240 Ser Glu Asn Val Ala Pro Phe Trp ProAla Val Ile Arg Thr Ala Leu 245 250 255 Thr Trp Lys Gly Leu Val Ser LeuLeu Arg Ser Gly Met Lys Ser Ile 260 265 270 Lys Gly Ala Leu Thr Met ProLeu Met Ile Glu Gly Tyr Lys Lys Gly 275 280 285 Val Ile Lys Phe Gly IleIle Thr Cys Gln Lys Pro Leu 290 295 300 34 301 PRT Brassica napus 34 MetVal Ala Val Thr Ala Ala Ala Thr Ser Ser Ala Glu Ala Leu Arg 1 5 10 15Glu Gly Ile Ala Glu Phe Tyr Asn Glu Thr Ser Gly Leu Trp Glu Glu 20 25 30Ile Trp Gly Asp His Met His His Gly Phe Tyr Asp Pro Asp Ser Ser 35 40 45Val Gln Leu Ser Asp Ser Gly His Arg Glu Ala Gln Ile Arg Met Ile 50 55 60Glu Glu Ser Leu Arg Phe Ala Gly Val Thr Glu Glu Glu Lys Lys Ile 65 70 7580 Lys Arg Val Val Asp Val Gly Cys Gly Ile Gly Gly Ser Ser Arg Tyr 85 9095 Ile Ala Ser Lys Phe Gly Ala Glu Cys Ile Gly Ile Thr Leu Ser Pro 100105 110 Val Gln Ala Lys Arg Ala Asn Asp Leu Ala Thr Ala Gln Ser Leu Ser115 120 125 His Lys Val Ser Phe Gln Val Ala Asp Ala Leu Asp Gln Pro PheGlu 130 135 140 Asp Gly Ile Ser Asp Leu Val Trp Ser Met Glu Ser Gly GluHis Met 145 150 155 160 Pro Asp Lys Ala Lys Phe Val Lys Glu Leu Val ArgVal Thr Ala Pro 165 170 175 Gly Gly Arg Ile Ile Ile Val Thr Trp Cys HisArg Asn Leu Ser Gln 180 185 190 Gly Glu Glu Ser Leu Gln Pro Trp Glu GlnAsn Leu Leu Asp Arg Ile 195 200 205 Cys Lys Thr Phe Tyr Leu Pro Ala TrpCys Ser Thr Thr Asp Tyr Val 210 215 220 Glu Leu Leu Gln Ser Leu Ser LeuGln Asp Ile Lys Tyr Ala Asp Trp 225 230 235 240 Ser Glu Asn Val Ala ProPhe Trp Pro Ala Val Ile Arg Thr Ala Leu 245 250 255 Thr Trp Lys Gly LeuVal Ser Leu Leu Arg Ser Gly Met Lys Ser Ile 260 265 270 Lys Gly Ala LeuThr Met Pro Leu Met Ile Glu Gly Tyr Lys Lys Gly 275 280 285 Val Ile LysPhe Gly Ile Ile Thr Cys Gln Lys Pro Leu 290 295 300 35 315 PRT Cupheapulcherrima 35 Met Ala Thr Ala Asp Lys Thr Gln Ser Thr Asp Thr Ser AsnGlu Gly 1 5 10 15 Val Val Ser Tyr Asp Thr Gln Val Leu Gln Lys Gly IleAla Glu Phe 20 25 30 Tyr Asp Glu Ser Ser Gly Ile Trp Glu Asp Ile Trp GlyAsp His Met 35 40 45 His His Gly Tyr Tyr Asp Gly Ser Thr Pro Val Ser LeuPro Asp His 50 55 60 Arg Ser Ala Gln Ile Arg Met Ile Asp Glu Ala Leu ArgPhe Ala Ser 65 70 75 80 Val Pro Ser Gly Glu Glu Asp Glu Ser Lys Ser LysIle Pro Lys Arg 85 90 95 Ile Val Asp Val Gly Cys Gly Ile Gly Gly Ser SerArg Tyr Leu Ala 100 105 110 Arg Lys Tyr Gly Ala Glu Cys Arg Gly Ile ThrLeu Ser Pro Val Gln 115 120 125 Ala Glu Arg Gly Asn Ser Leu Ala Arg SerGln Gly Leu Ser Asp Lys 130 135 140 Val Ser Phe Gln Val Ala Asp Ala LeuAla Gln Pro Phe Pro Asp Gly 145 150 155 160 Gln Phe Asp Leu Val Trp SerMet Glu Ser Gly Glu His Met Pro Asp 165 170 175 Lys Ser Lys Phe Val AsnGlu Leu Val Arg Val Ala Ala Pro Gly Gly 180 185 190 Thr Ile Ile Ile ValThr Trp Cys His Arg Asp Leu Arg Glu Asp Glu 195 200 205 Asp Ala Leu GlnPro Arg Glu Lys Glu Ile Leu Asp Lys Ile Cys Asn 210 215 220 Pro Phe TyrLeu Pro Ala Trp Cys Ser Ala Ala Asp Tyr Val Lys Leu 225 230 235 240 LeuGln Ser Leu Asp Val Glu Asp Ile Lys Ser Ala Asp Trp Thr Pro 245 250 255Tyr Val Ala Pro Phe Trp Pro Ala Val Leu Lys Ser Ala Phe Thr Ile 260 265270 Lys Gly Phe Val Ser Leu Leu Arg Ser Gly Met Lys Thr Ile Lys Gly 275280 285 Ala Phe Ala Met Pro Leu Met Ile Glu Gly Tyr Lys Lys Gly Val Ile290 295 300 Lys Phe Ser Ile Ile Thr Cys Arg Lys Pro Glu 305 310 315 36299 PRT Gossypium hirsutum 36 Met Val Lys Ala Ala Ala Ser Ser Leu SerThr Thr Thr Leu Gln Glu 1 5 10 15 Gly Ile Ala Glu Phe Tyr Asp Glu SerSer Gly Ile Trp Glu Asp Ile 20 25 30 Trp Gly Asp His Met His His Gly TyrTyr Glu Pro Gly Ser Asp Ile 35 40 45 Ser Gly Ser Asp His Arg Ala Ala GlnIle Arg Met Val Glu Glu Ser 50 55 60 Leu Arg Phe Ala Gly Ile Ser Glu AspPro Ala Asn Arg Pro Lys Arg 65 70 75 80 Ile Val Asp Val Gly Cys Gly IleGly Gly Ser Ser Arg Tyr Leu Ala 85 90 95 Arg Lys Tyr Gly Ala Lys Cys GlnGly Ile Thr Leu Ser Pro Val Gln 100 105 110 Ala Gly Arg Ala Asn Ala LeuAla Asn Ala Gln Gly Leu Ala Glu Gln 115 120 125 Val Cys Phe Glu Val AlaAsp Ala Leu Asn Gln Pro Phe Pro Asp Asp 130 135 140 Gln Phe Asp Leu ValTrp Ser Met Glu Ser Gly Glu His Met Pro Asp 145 150 155 160 Lys Pro LysPhe Val Lys Glu Leu Val Val Ala Ala Pro Gly Gly Thr 165 170 175 Ile IleVal Val Thr Trp Cys His Arg Asp Leu Gly Pro Ser Glu Glu 180 185 190 SerLeu Gln Pro Trp Glu Gln Lys Leu Leu Asn Arg Ile Cys Asp Ala 195 200 205Tyr Tyr Leu Pro Glu Trp Cys Ser Thr Ser Asp Tyr Val Lys Leu Phe 210 215220 Gln Ser Leu Ser Leu Gln Asp Ile Lys Ala Gly Asp Trp Thr Glu Asn 225230 235 240 Val Ala Pro Phe Trp Pro Ala Val Ile Arg Ser Ala Leu Thr TrpLys 245 250 255 Gly Phe Thr Ser Leu Leu Arg Ser Gly Leu Lys Thr Ile LysGly Ala 260 265 270 Leu Val Met Pro Leu Met Ile Glu Gly Phe Gln Lys GlyVal Ile Lys 275 280 285 Phe Ala Ile Ile Ala Cys Arg Lys Pro Ala Glu 290295 37 311 PRT Tagetes erecta 37 Met Ala Leu Ser Val Val Ala Ala Glu ValPro Val Thr Val Thr Pro 1 5 10 15 Ala Thr Thr Lys Ala Glu Asp Val GluLeu Lys Lys Gly Ile Ala Glu 20 25 30 Phe Tyr Asp Glu Ser Ser Glu Met TrpGlu Asn Ile Trp Gly Glu His 35 40 45 Met His His Gly Tyr Tyr Asn Thr AsnAla Val Val Glu Leu Ser Asp 50 55 60 His Arg Ser Ala Gln Ile Arg Met IleGlu Gln Ala Leu Leu Phe Ala 65 70 75 80 Ser Val Ser Asp Asp Pro Val LysLys Pro Arg Ser Ile Val Asp Val 85 90 95 Gly Cys Gly Ile Gly Gly Ser SerArg Tyr Leu Ala Lys Lys Tyr Glu 100 105 110 Ala Glu Cys His Gly Ile ThrLeu Ser Pro Val Gln Ala Glu Arg Ala 115 120 125 Gln Ala Leu Ala Ala AlaGln Gly Leu Ala Asp Lys Ala Ser Phe Gln 130 135 140 Val Ala Asp Ala LeuAsp Gln Pro Phe Pro Asp Gly Lys Phe Asp Leu 145 150 155 160 Val Trp SerMet Glu Ser Gly Glu His Met Pro Asp Lys Leu Lys Phe 165 170 175 Val SerGlu Leu Val Arg Val Ala Ala Pro Gly Ala Thr Ile Ile Ile 180 185 190 ValThr Trp Cys His Arg Asp Leu Ser Pro Gly Glu Lys Ser Leu Arg 195 200 205Pro Asp Glu Glu Lys Ile Leu Lys Lys Ile Cys Ser Ser Phe Tyr Leu 210 215220 Pro Ala Trp Cys Ser Thr Ser Asp Tyr Val Lys Leu Leu Glu Ser Leu 225230 235 240 Ser Leu Gln Asp Ile Lys Ala Ala Asp Trp Ser Ala Asn Val AlaPro 245 250 255 Phe Trp Pro Ala Val Ile Lys Thr Ala Leu Ser Trp Lys GlyIle Thr 260 265 270 Ser Leu Leu Arg Ser Gly Trp Lys Ser Ile Arg Gly AlaMet Val Met 275 280 285 Pro Leu Met Ile Glu Gly Phe Lys Lys Asp Ile IleLys Phe Ser Ile 290 295 300 Ile Thr Cys Lys Lys Pro Glu 305 310 38 305PRT Zea mays 38 Met Ala Ser Ser Thr Ala Gln Ala Pro Ala Thr Ala Pro ProGly Leu 1 5 10 15 Lys Glu Gly Ile Ala Gly Leu Tyr Asp Glu Ser Ser GlyLeu Trp Glu 20 25 30 Asn Ile Trp Gly Asp His Met His His Gly Phe Tyr AspSer Ser Glu 35 40 45 Ala Ala Ser Met Ala Asp His Arg Arg Ala Gln Ile ArgMet Ile Glu 50 55 60 Glu Ala Leu Ala Phe Ala Gly Val Pro Ala Ser Asp AspPro Glu Lys 65 70 75 80 Thr Pro Lys Thr Ile Val Asp Val Gly Cys Gly IleGly Gly Ser Ser 85 90 95 Arg Tyr Leu Ala Lys Lys Tyr Gly Ala Gln Cys ThrGly Ile Thr Leu 100 105 110 Ser Pro Val Gln Ala Glu Arg Gly Asn Ala LeuAla Ala Ala Gln Gly 115 120 125 Leu Ser Asp Gln Val Thr Leu Gln Val AlaAsp Ala Leu Glu Gln Pro 130 135 140 Phe Pro Asp Gly Gln Phe Asp Leu ValTrp Ser Met Glu Ser Gly Glu 145 150 155 160 His Met Pro Asp Lys Arg LysPhe Val Ser Glu Leu Ala Arg Val Ala 165 170 175 Ala Pro Gly Gly Thr IleIle Ile Val Thr Trp Cys His Arg Asn Leu 180 185 190 Asp Pro Ser Glu ThrSer Leu Lys Pro Asp Glu Leu Ser Leu Leu Arg 195 200 205 Arg Ile Cys AspAla Tyr Tyr Leu Pro Asp Trp Cys Ser Pro Ser Asp 210 215 220 Tyr Val AsnIle Ala Lys Ser Leu Ser Leu Glu Asp Ile Lys Thr Ala 225 230 235 240 AspTrp Ser Glu Asn Val Ala Pro Phe Trp Pro Ala Val Ile Lys Ser 245 250 255Ala Leu Thr Trp Lys Gly Phe Thr Ser Leu Leu Thr Thr Gly Trp Lys 260 265270 Thr Ile Arg Gly Ala Met Val Met Pro Leu Met Ile Gln Gly Tyr Lys 275280 285 Lys Gly Leu Ile Lys Phe Thr Ile Ile Thr Cys Arg Lys Pro Gly Ala290 295 300 Ala 305 39 280 PRT Nostoc punctiforme 39 Met Ser Ala Thr LeuTyr Gln Gln Ile Gln Gln Phe Tyr Asp Ala Ser 1 5 10 15 Ser Gly Leu TrpGlu Gln Ile Trp Gly Glu His Met His His Gly Tyr 20 25 30 Tyr Gly Ala AspGly Thr Gln Lys Lys Asp Arg Arg Gln Ala Gln Ile 35 40 45 Asp Leu Ile GluGlu Leu Leu Asn Trp Ala Gly Val Gln Ala Ala Glu 50 55 60 Asp Ile Leu AspVal Gly Cys Gly Ile Gly Gly Ser Ser Leu Tyr Leu 65 70 75 80 Ala Gln LysPhe Asn Ala Lys Ala Thr Gly Ile Thr Leu Ser Pro Val 85 90 95 Gln Ala AlaArg Ala Thr Glu Arg Ala Leu Glu Ala Asn Leu Ser Leu 100 105 110 Arg ThrGln Phe Gln Val Ala Asn Ala Gln Ala Met Pro Phe Ala Asp 115 120 125 AspSer Phe Asp Leu Val Trp Ser Leu Glu Ser Gly Glu His Met Pro 130 135 140Asp Lys Thr Lys Phe Leu Gln Glu Cys Tyr Arg Val Leu Lys Pro Gly 145 150155 160 Gly Lys Leu Ile Met Val Thr Trp Cys His Arg Pro Thr Asp Glu Ser165 170 175 Pro Leu Thr Ala Asp Glu Glu Lys His Leu Gln Asp Ile Tyr ArgVal 180 185 190 Tyr Cys Leu Pro Tyr Val Ile Ser Leu Pro Glu Tyr Glu AlaIle Ala 195 200 205 His Gln Leu Pro Leu His Asn Ile Arg Thr Ala Asp TrpSer Thr Ala 210 215 220 Val Ala Pro Phe Trp Asn Val Val Ile Asp Ser AlaPhe Thr Pro Gln 225 230 235 240 Ala Leu Trp Gly Leu Leu Asn Ala Gly TrpThr Thr Ile Gln Gly Ala 245 250 255 Leu Ser Leu Gly Leu Met Arg Arg GlyTyr Glu Arg Gly Leu Ile Arg 260 265 270 Phe Gly Leu Leu Cys Gly Asn Lys275 280 40 280 PRT Anabaena sp. 40 Met Ser Ala Thr Leu Tyr Gln Gln IleGln Gln Phe Tyr Asp Ala Ser 1 5 10 15 Ser Gly Leu Trp Glu Glu Ile TrpGly Glu His Met His His Gly Tyr 20 25 30 Tyr Gly Ala Asp Gly Thr Glu GlnLys Asn Arg Arg Gln Ala Gln Ile 35 40 45 Asp Leu Ile Glu Glu Leu Leu ThrTrp Ala Gly Val Gln Thr Ala Glu 50 55 60 Asn Ile Leu Asp Val Gly Cys GlyIle Gly Gly Ser Ser Leu Tyr Leu 65 70 75 80 Ala Gly Lys Leu Asn Ala LysAla Thr Gly Ile Thr Leu Ser Pro Val 85 90 95 Gln Ala Ala Arg Ala Thr GluArg Ala Lys Glu Ala Gly Leu Ser Gly 100 105 110 Arg Ser Gln Phe Leu ValAla Asn Ala Gln Ala Met Pro Phe Asp Asp 115 120 125 Asn Ser Phe Asp LeuVal Trp Ser Leu Glu Ser Gly Glu His Met Pro 130 135 140 Asp Lys Thr LysPhe Leu Gln Glu Cys Tyr Arg Val Leu Lys Pro Gly 145 150 155 160 Gly LysLeu Ile Met Val Thr Trp Cys His Arg Pro Thr Asp Lys Thr 165 170 175 ProLeu Thr Ala Asp Glu Lys Lys His Leu Glu Asp Ile Tyr Arg Val 180 185 190Tyr Cys Leu Pro Tyr Val Ile Ser Leu Pro Glu Tyr Glu Ala Ile Ala 195 200205 Arg Gln Leu Pro Leu Asn Asn Ile Arg Thr Ala Asp Trp Ser Gln Ser 210215 220 Val Ala Gln Phe Trp Asn Ile Val Ile Asp Ser Ala Phe Thr Pro Gln225 230 235 240 Ala Ile Phe Gly Leu Leu Arg Ala Gly Trp Thr Thr Ile GlnGly Ala 245 250 255 Leu Ser Leu Gly Leu Met Arg Arg Gly Tyr Glu Arg GlyLeu Ile Arg 260 265 270 Phe Gly Leu Leu Cys Gly Asp Lys 275 280 41 317PRT Synechocystis PCC 6803 41 Met Val Tyr His Val Arg Pro Lys His AlaLeu Phe Leu Ala Phe Tyr 1 5 10 15 Cys Tyr Phe Ser Leu Leu Thr Met AlaSer Ala Thr Ile Ala Ser Ala 20 25 30 Asp Leu Tyr Glu Lys Ile Lys Asn PheTyr Asp Asp Ser Ser Gly Leu 35 40 45 Trp Glu Asp Val Trp Gly Glu His MetHis His Gly Tyr Tyr Gly Pro 50 55 60 His Gly Thr Tyr Arg Ile Asp Arg ArgGln Ala Gln Ile Asp Leu Ile 65 70 75 80 Lys Glu Leu Leu Ala Trp Ala ValPro Gln Asn Ser Ala Lys Pro Arg 85 90 95 Lys Ile Leu Asp Leu Gly Cys GlyIle Gly Gly Ser Ser Leu Tyr Leu 100 105 110 Ala Gln Gln His Gln Ala GluVal Met Gly Ala Ser Leu Ser Pro Val 115 120 125 Gln Val Glu Arg Ala GlyGlu Arg Ala Arg Ala Leu Gly Leu Gly Ser 130 135 140 Thr Cys Gln Phe GlnVal Ala Asn Ala Leu Asp Leu Pro Phe Ala Ser 145 150 155 160 Asp Ser PheAsp Trp Val Trp Ser Leu Glu Ser Gly Glu His Met Pro 165 170 175 Asn LysAla Gln Phe Leu Gln Glu Ala Trp Arg Val Leu Lys Pro Gly 180 185 190 GlyArg Leu Ile Leu Ala Thr Trp Cys His Arg Pro Ile Asp Pro Gly 195 200 205Asn Gly Pro Leu Thr Ala Asp Glu Arg Arg His Leu Gln Ala Ile Tyr 210 215220 Asp Val Tyr Cys Leu Pro Tyr Val Val Ser Leu Pro Asp Tyr Glu Ala 225230 235 240 Ile Ala Arg Glu Cys Gly Phe Gly Glu Ile Lys Thr Ala Asp TrpSer 245 250 255 Val Ala Val Ala Pro Phe Trp Asp Arg Val Ile Glu Ser AlaPhe Asp 260 265 270 Pro Arg Val Leu Trp Ala Leu Gly Gln Ala Gly Pro LysIle Ile Asn 275 280 285 Ala Ala Leu Cys Leu Arg Leu Met Lys Trp Gly TyrGlu Arg Gly Leu 290 295 300 Val Arg Phe Gly Leu Leu Thr Gly Ile Lys ProLeu Val 305 310 315 42 957 DNA Synechocystis PCC 6803 42 atgcccgagtatttgcttct gcccgctggc ctaatttccc tctccctggc gatcgccgct 60 ggactgtatctcctaactgc ccggggctat cagtcatcgg attccgtggc caacgcctac 120 gaccaatggacagaggacgg cattttggaa tattactggg gcgaccatat ccacctcggc 180 cattatggcgatccgccagt ggccaaggat ttcatccaat cgaaaattga ttttgtccat 240 gccatggcccagtggggcgg attagataca cttccccccg gcacaacggt attggatgtg 300 ggttgcggcattggcggtag cagtcgcatt ctcgccaaag attatggttt taacgttacc 360 ggcatcaccattagtcccca acaggtgaaa cgggcgacgg aattaactcc tcccgatgtg 420 acggccaagtttgcggtgga cgatgctatg gctttgtctt ttcctgacgg tagtttcgac 480 gtagtttggtcggtggaagc agggccccac atgcctgaca aagctgtgtt tgccaaggaa 540 ttactgcgggtcgtgaaacc agggggcatt ctggtggtgg cggattggaa tcaacgggac 600 gatcgccaagtgcccctcaa cttctgggaa aaaccagtga tgcgacaact gttggatcaa 660 tggtcccaccctgcctttgc cagcattgaa ggttttgcgg aaaatttgga agccacgggt 720 ttggtggagggccaggtgac tactgctgat tggactgtac cgaccctccc cgcttggttg 780 gataccatttggcagggcat tatccggccc cagggctggt tacaatacgg cattcgtggg 840 tttatcaaatccgtgcggga agtaccgact attttattga tgcgccttgc ctttggggta 900 ggactttgtcgcttcggtat gttcaaagca gtgcgaaaaa acgccactca agcttaa 957 43 993 DNAAnabaena sp. 43 atgagttggt tgttttctac actggtattt ttcttaacgc tattgacagcagggatcgcg 60 ttatatctca ttactgctag acgttatcaa tcatctaact ccgtagccaattcctacgac 120 cagtggactg aagacggtat tttagagttt tactggggcg aacatatccatttaggtcat 180 tatggttcgc cacctcaaag aaaggatttt ctggtggcta aatctgattttgtccatgaa 240 atggtgcgtt ggggtggttt ggataaacta ccccctggta ctaccttgttagatgttggt 300 tgtggaattg ggggtagtag tcgcattttg gcacgggatt atggatttgccgttacaggt 360 atcaccatca gcccccaaca agtccaacgc gctcaagagt taacaccacaggaactgaat 420 gcacagtttt tggtggatga tgcaatggcg ctttccttcc cagataatagttttgatgta 480 gtttggtcaa ttgaagctgg cccacatatg ccagataaag ccatttttgccaaagaattg 540 atgcgggtac taaagcctgg tggaatcatg gttttagccg actggaatcagcgagacgat 600 cgccaaaaac ccctcaattt ttgggagaaa ccagtaatgc agcaactactagatcagtgg 660 tctcatccag ctttttccag catcgaaggc ttttctgagc ttttggcagcgacgggatta 720 gtagaagggg aggtaatcac cgcagactgg acgaaacaaa cactcccctcttggcttgat 780 tctatctggc aaggaatagt tagaccagaa ggattagtgc gttttggtctatctggtttc 840 attaaatctc tgcgagaagt gcctacccta ctactgatga ggctggcattcggtacagga 900 ctctgtagat ttgggatgtt ccgcgcttta cgagctgaca ctgtaagatcatcagcagaa 960 cagacatctg cgatcaaggt tgctcaaaag taa 993 44 930 DNASynechococcus sp. MT1 44 atgttggctg gcctgcttct cctgaccggg gctgccggtgccacggccct gctgatctgg 60 ttgcagcgtg atcgccgcta ccactcctca gacagcgtcgccgcggccta cgacgcctgg 120 accgatgacc aactgctgga acggctctgg ggagaccatgtccacctggg gcattacgga 180 aacccgccag gttctgtcga cttccgccag gccaaggaggcttttgtgca cgagctggtg 240 cgctggagcg ggctcgacca actacctcga ggcagtcgggtgttggatgt gggttgcggc 300 atcggcggca gtgcccggat cctggccagg gattacggcttggacgtgct cggggtgagc 360 atcagcccag cccagatccg ccgcgccaca gaactcacccccgccggcct cagctgtcgc 420 tttgaagtga tggacgccct taaccttcaa cttcccgatcggcaattcga tgcggtgtgg 480 acggtggagg cggggcccca catgccagac aagcagcgtttcgctgatga gttgctgcgg 540 gtactccggc ccgggggctg cttagccgcc gctgattggaaccgccgcgc ccccaaggat 600 ggcgccatga acagcaccga acgctgggtg atgcggcagttgttgaatca atgggcgcat 660 ccggaattcg ccagcatctc cggcttccgg gccaacctcgaagccagccc tcaccagcgg 720 ggcctgatca gtaccggcga ctggactctg gccacccttccctcctggtt tgattcgatc 780 gccgaaggcc tccgtcgccc ctgggctgtc ctgggccttggtcccaaagc agtgcttcaa 840 ggcctgcgag agaccccgac gctgctgttg atgcattgggcctttgccac agggttgatg 900 cagttcggcg tctttcgcct cagccgctga 930 45 936DNA Prochlorococcus marinus 45 atgtccattt ttttaatatc ttcacttgttatatttttaa ctttattatt ttcttctcta 60 atactttgga gaattaatac tagaaaatatatttcttcga gaactgtagc tacagcatat 120 gattcctgga ctcaagataa attactagaaagattatggg gagaacatat acatctaggt 180 ttctatcctc taaataaaaa tattgattttagagaggcta aagttcaatt tgtacatgag 240 ttagtaagtt ggagtggttt agataaattaccaagaggtt ctaggatttt agatgtcggt 300 tgcggaatag gtggaagttc tagaattctcgccaattatt atggatttaa tgtcactgga 360 ataactatta gtccagctca agtaaaaagagcaaaagaac ttactcctta tgaatgtaaa 420 tgcaacttca aagttatgga tgctttggatttgaaatttg aagagggaat atttgatggt 480 gtttggagtg ttgaggcagg agcccatatgaataataaaa ctaaatttgc agatcaaatg 540 ttaagaactt taagacctgg aggatatttagcattggctg attggaattc aagagattta 600 caaaagcaac ccccatccat gattgaaaaaataatcttaa aacaattact tgaacagtgg 660 gtacatccta aatttattag tatcaatgaattcagtagta ttcttataaa taacaaaaat 720 agttcaggtc aagttatatc ctctaattggaattctttta caaatccctc ttggtttgat 780 tcaatatttg aaggaatgag aagacctaattcaattttat cccttggtcc aggagcaatt 840 ataaagtcta tcagagagat acctacaatacttttaatgg attgggcctt taaaaaaggt 900 ttaatggaat ttggagttta taaatgtagaggttaa 936 46 318 PRT Synechocystis PCC6803 46 Met Pro Glu Tyr Leu LeuLeu Pro Ala Gly Leu Ile Ser Leu Ser Leu 1 5 10 15 Ala Ile Ala Ala GlyLeu Tyr Leu Leu Thr Ala Arg Gly Tyr Gln Ser 20 25 30 Ser Asp Ser Val AlaAsn Ala Tyr Asp Gln Trp Thr Glu Asp Gly Ile 35 40 45 Leu Glu Tyr Tyr TrpGly Asp His Ile His Leu Gly His Tyr Gly Asp 50 55 60 Pro Pro Val Ala LysAsp Phe Ile Gln Ser Lys Ile Asp Phe Val His 65 70 75 80 Ala Met Ala GlnTrp Gly Gly Leu Asp Thr Leu Pro Pro Gly Thr Thr 85 90 95 Val Leu Asp ValGly Cys Gly Ile Gly Gly Ser Ser Arg Ile Leu Ala 100 105 110 Lys Asp TyrGly Phe Asn Val Thr Gly Ile Thr Ile Ser Pro Gln Gln 115 120 125 Val LysArg Ala Thr Glu Leu Thr Pro Pro Asp Val Thr Ala Lys Phe 130 135 140 AlaVal Asp Asp Ala Met Ala Leu Ser Phe Pro Asp Gly Ser Phe Asp 145 150 155160 Val Val Trp Ser Val Glu Ala Gly Pro His Met Pro Asp Lys Ala Val 165170 175 Phe Ala Lys Glu Leu Leu Arg Val Val Lys Pro Gly Gly Ile Leu Val180 185 190 Val Ala Asp Trp Asn Gln Arg Asp Asp Arg Gln Val Pro Leu AsnPhe 195 200 205 Trp Glu Lys Pro Val Met Arg Gln Leu Leu Asp Gln Trp SerHis Pro 210 215 220 Ala Phe Ala Ser Ile Glu Gly Phe Ala Glu Asn Leu GluAla Thr Gly 225 230 235 240 Leu Val Glu Gly Gln Val Thr Thr Ala Asp TrpThr Val Pro Thr Leu 245 250 255 Pro Ala Trp Leu Asp Thr Ile Trp Gln GlyIle Ile Arg Pro Gln Gly 260 265 270 Trp Leu Gln Tyr Gly Ile Arg Gly PheIle Lys Ser Val Arg Glu Val 275 280 285 Pro Thr Ile Leu Leu Met Arg LeuAla Phe Gly Val Gly Leu Cys Arg 290 295 300 Phe Gly Met Phe Lys Ala ValArg Lys Asn Ala Thr Gln Ala 305 310 315 47 330 PRT Anabaena sp. 47 MetSer Trp Leu Phe Ser Thr Leu Val Phe Phe Leu Thr Leu Leu Thr 1 5 10 15Ala Gly Ile Ala Leu Tyr Leu Ile Thr Ala Arg Arg Tyr Gln Ser Ser 20 25 30Asn Ser Val Ala Asn Ser Tyr Asp Gln Trp Thr Glu Asp Gly Ile Leu 35 40 45Glu Phe Tyr Trp Gly Glu His Ile His Leu Gly His Tyr Gly Ser Pro 50 55 60Pro Gln Arg Lys Asp Phe Leu Val Ala Lys Ser Asp Phe Val His Glu 65 70 7580 Met Val Arg Trp Gly Gly Leu Asp Lys Leu Pro Pro Gly Thr Thr Leu 85 9095 Leu Asp Val Gly Cys Gly Ile Gly Gly Ser Ser Arg Ile Leu Ala Arg 100105 110 Asp Tyr Gly Phe Ala Val Thr Gly Ile Thr Ile Ser Pro Gln Gln Val115 120 125 Gln Arg Ala Gln Glu Leu Thr Pro Gln Glu Leu Asn Ala Gln PheLeu 130 135 140 Val Asp Asp Ala Met Ala Leu Ser Phe Pro Asp Asn Ser PheAsp Val 145 150 155 160 Val Trp Ser Ile Glu Ala Gly Pro His Met Pro AspLys Ala Ile Phe 165 170 175 Ala Lys Glu Leu Met Arg Val Leu Lys Pro GlyGly Ile Met Val Leu 180 185 190 Ala Asp Trp Asn Gln Arg Asp Asp Arg GlnLys Pro Leu Asn Phe Trp 195 200 205 Glu Lys Pro Val Met Gln Gln Leu LeuAsp Gln Trp Ser His Pro Ala 210 215 220 Phe Ser Ser Ile Glu Gly Phe SerGlu Leu Leu Ala Ala Thr Gly Leu 225 230 235 240 Val Glu Gly Glu Val IleThr Ala Asp Trp Thr Lys Gln Thr Leu Pro 245 250 255 Ser Trp Leu Asp SerIle Trp Gln Gly Ile Val Arg Pro Glu Gly Leu 260 265 270 Val Arg Phe GlyLeu Ser Gly Phe Ile Lys Ser Leu Arg Glu Val Pro 275 280 285 Thr Leu LeuLeu Met Arg Leu Ala Phe Gly Thr Gly Leu Cys Arg Phe 290 295 300 Gly MetPhe Arg Ala Leu Arg Ala Asp Thr Val Arg Ser Ser Ala Glu 305 310 315 320Gln Thr Ser Ala Ile Lys Val Ala Gln Lys 325 330 48 309 PRT Synechococcussp. 48 Met Leu Ala Gly Leu Leu Leu Leu Thr Gly Ala Ala Gly Ala Thr Ala 15 10 15 Leu Leu Ile Trp Leu Gln Arg Asp Arg Arg Tyr His Ser Ser Asp Ser20 25 30 Val Ala Ala Ala Tyr Asp Ala Trp Thr Asp Asp Gln Leu Leu Glu Arg35 40 45 Leu Trp Gly Asp His Val His Leu Gly His Tyr Gly Asn Pro Pro Gly50 55 60 Ser Val Asp Phe Arg Gln Ala Lys Glu Ala Phe Val His Glu Leu Val65 70 75 80 Arg Trp Ser Gly Leu Asp Gln Leu Pro Arg Gly Ser Arg Val LeuAsp 85 90 95 Val Gly Cys Gly Ile Gly Gly Ser Ala Arg Ile Leu Ala Arg AspTyr 100 105 110 Gly Leu Asp Val Leu Gly Val Ser Ile Ser Pro Ala Gln IleArg Arg 115 120 125 Ala Thr Glu Leu Thr Pro Ala Gly Leu Ser Cys Arg PheGlu Val Met 130 135 140 Asp Ala Leu Asn Leu Gln Leu Pro Asp Arg Gln PheAsp Ala Val Trp 145 150 155 160 Thr Val Glu Ala Gly Pro His Met Pro AspLys Gln Arg Phe Ala Asp 165 170 175 Glu Leu Leu Arg Val Leu Arg Pro GlyGly Cys Leu Ala Ala Ala Asp 180 185 190 Trp Asn Arg Arg Ala Pro Lys AspGly Ala Met Asn Ser Thr Glu Arg 195 200 205 Trp Val Met Arg Gln Leu LeuAsn Gln Trp Ala His Pro Glu Phe Ala 210 215 220 Ser Ile Ser Gly Phe ArgAla Asn Leu Glu Ala Ser Pro His Gln Arg 225 230 235 240 Gly Leu Ile SerThr Gly Asp Trp Thr Leu Ala Thr Leu Pro Ser Trp 245 250 255 Phe Asp SerIle Ala Glu Gly Leu Arg Arg Pro Trp Ala Val Leu Gly 260 265 270 Leu GlyPro Lys Ala Val Leu Gln Gly Leu Arg Glu Thr Pro Thr Leu 275 280 285 LeuLeu Met His Trp Ala Phe Ala Thr Gly Leu Met Gln Phe Gly Val 290 295 300Phe Arg Leu Ser Arg 305 49 311 PRT Prochlorococcus marinus 49 Met SerIle Phe Leu Ile Ser Ser Leu Val Ile Phe Leu Thr Leu Leu 1 5 10 15 PheSer Ser Leu Ile Leu Trp Arg Ile Asn Thr Arg Lys Tyr Ile Ser 20 25 30 SerArg Thr Val Ala Thr Ala Tyr Asp Ser Trp Thr Gln Asp Lys Leu 35 40 45 LeuGlu Arg Leu Trp Gly Glu His Ile His Leu Gly Phe Tyr Pro Leu 50 55 60 AsnLys Asn Ile Asp Phe Arg Glu Ala Lys Val Gln Phe Val His Glu 65 70 75 80Leu Val Ser Trp Ser Gly Leu Asp Lys Leu Pro Arg Gly Ser Arg Ile 85 90 95Leu Asp Val Gly Cys Gly Ile Gly Gly Ser Ser Arg Ile Leu Ala Asn 100 105110 Tyr Tyr Gly Phe Asn Val Thr Gly Ile Thr Ile Ser Pro Ala Gln Val 115120 125 Lys Arg Ala Lys Glu Leu Thr Pro Tyr Glu Cys Lys Cys Asn Phe Lys130 135 140 Val Met Asp Ala Leu Asp Leu Lys Phe Glu Glu Gly Ile Phe AspGly 145 150 155 160 Val Trp Ser Val Glu Ala Gly Ala His Met Asn Asn LysThr Lys Phe 165 170 175 Ala Asp Gln Met Leu Arg Thr Leu Arg Pro Gly GlyTyr Leu Ala Leu 180 185 190 Ala Asp Trp Asn Ser Arg Asp Leu Gln Lys GlnPro Pro Ser Met Ile 195 200 205 Glu Lys Ile Ile Leu Lys Gln Leu Leu GluGln Trp Val His Pro Lys 210 215 220 Phe Ile Ser Ile Asn Glu Phe Ser SerIle Leu Ile Asn Asn Lys Asn 225 230 235 240 Ser Ser Gly Gln Val Ile SerSer Asn Trp Asn Ser Phe Thr Asn Pro 245 250 255 Ser Trp Phe Asp Ser IlePhe Glu Gly Met Arg Arg Pro Asn Ser Ile 260 265 270 Leu Ser Leu Gly ProGly Ala Ile Ile Lys Ser Ile Arg Glu Ile Pro 275 280 285 Thr Ile Leu LeuMet Asp Trp Ala Phe Lys Lys Gly Leu Met Glu Phe 290 295 300 Gly Val TyrLys Cys Arg Gly 305 310 50 6864 DNA Oryza sativa 50 cgaggcccccgtccagctgc catgtggcgg ggacagcaag cgggaagggg acccaggctg 60 aaccgctatcaatgcgcgcg gcgccccaac tgcccctcgc cgcattaaat gcggtagggg 120 cagacatgaggtgccgcccg actgacgcac atcagtcagg agtgaccggt ctgtgaccgg 180 cctatcgccggtcacgtctg actggacggg cggccgtgcc cccacgtcgc ctctgtcccc 240 ggcggagtggaggtaggtat gacccgtccc atcggagcgt gattcggagc tccctccacg 300 agaaggacgtccaccacaag tcttcaccca tttatgaggg aatgacaggg ctgtcccccg 360 tgtcaggcagggggcgacga tgggtcccgc ttaaggacga gcggtggctt ggcttccggg 420 cgaaggcacgaattgtgatc tggtcaaggt agggtgggtc cccaccctgt agaagagtag 480 gtagaggcggtgcatgtggt ttccccttga gctataaaag gaggacctta cccaccgaga 540 aagacgacgactctcaggaa gcctgagctc taggagaaga gaagcgagaa cgctctccgg 600 agtttaggaacccttgtaac tctcaacctt aaatcccaca cacagaagta gggtattacg 660 ctccatcgcggcccgaacct gtataattct cttgcccata cgcgactagc aagacttagg 720 gcggatacgcgatctctaga ggcgagccct tttccctagc cgaactcaca aaaggggatc 780 tcacgatctcccgatagaga ggattactcc tcgacaatgt cattttaata tatttgaata 840 atataatgagaaaacatata tgctattata tgagagaaaa tataatgatg ctagccgcgc 900 aatctgcacgggccatcatg ctagttttaa taataagaaa atagtacgag aaggttagta 960 aaaagtactgaatggataaa acttctcgat ttacatagca aatactagtt aaagggatat 1020 aagtgaacttattttaagat aactagcaca atatcaatgc gttgtaatgg atattataaa 1080 agaaaaaaggtaaaatttac aagtttttag cacaataaat ccatttggca tataatgcct 1140 acctggtgttaagatttaat gacacaatac gatcaatgta ttgataggca taaacttcga 1200 gtcaactaaaagagcttatt taaggggtgt caaacctatg aacccatacg tcacgaaggg 1260 ggtagtgctggcaaattcaa tgcccctacc attgctctct tttaaaatgg tatagactta 1320 tactatcatcatcacattct agtatgatgt tggtttattt tggtgtggtg tcgcgtgttc 1380 tctaaacgcactgtgtcgtg cttcatccat cttaaaataa tcttatcttt tacctatttc 1440 acacgtaccaataaaaatct ctaatttatt aaatgctaat ctcttcgtct caaagtgaac 1500 agaagaatatactagtaata caattatttc tcttctaatc cgctctcagt agatttaccc 1560 cgtacttacagccctctaca atcctcccta aacacagagt gctatagcac tgtgcagtgc 1620 agtgcctcattcgtctcaaa ataatcctat cttttaccta tttcacgtac caataaaaat 1680 atctaatttattaaatatta atatctccat ctcaaaataa acagaaaaat atactagtaa 1740 tacaattactacttatgttc caatccgctc tcagtcgact taccccgcac ttggcagtct 1800 ggcctctaccatccttgcgc cgtcgcgcgc tcgtgccggg gcttggctgc gagcgaataa 1860 aaagaaaagaaaacgtcaag gcctcagacg ccagagcgct acaaaatggc ccacgccgcc 1920 gcggccacgggcgcactggc accgctgcat ccactgctcc gctgcacgag ccgtcatctc 1980 tgcgcctcggcttcccctcg cgccggcctc tgcctccacc accaccgccg ccgccgccgc 2040 agcagccggaggacgaaact cgccgtgcgc gcgatggcac cgacgttgtc ctcgtcgtcg 2100 acggcggcggcagctccccc ggggctgaag gagggcatcg cggggctcta cgacgagtcg 2160 tccggcgtgtgggagagcat ctggggcgag cacatgcacc acggcttcta cgacgccggc 2220 gaggccgcctccatgtccga ccaccgccgc gcccagatcc gcatgatcga ggaatccctc 2280 gccttcgccgccgtccccgg tacgtattgc ccgccccact ccgccccccc ggaatctacg 2340 cttgcctggctgcgcggctg agcccatcca gcttttctgt ttggctgcaa gccgctggcc 2400 atagagaatcccggccattt ttctgctgct taacagttgt ttctgcctac ctaacccctg 2460 ctcgcctcacggctccagca cgtacggcaa aatcatgaga tattcagggg gttttctttt 2520 tctttttctttttggagacg ataaactcac catcttgatt ggtgtgggtt taattttgta 2580 cagtaccactattttgttcc attcattcat tcacttggta ctcgtactac gaaaggtgtg 2640 gacctgtgacagttttggca cgcactattc ccaacagggg ttttgcagtc ctatctcaca 2700 cggtagaccgtttgagcatt tagggccctt ttaaatcata gaaataaaaa aacaaaggaa 2760 taagaaaagcacaggattct aacaggaata caattgtaaa acagaggatt gcaaaacaca 2820 ggaaaaatacaggaatgacc gtttgattgg accacgagaa aaatgtagga atcatatgag 2880 agagatagactcaggaaatg ttccaagagg ttagacttct tgctaacttt cctccaaaat 2940 gtgcataggattacccattc cataggaatt ttaaaggatt gtatatgatt caatcctttg 3000 tttcaaagaccttcatatga tttttttttc cataggattg aaatcctcta aaatttctac 3060 atttttcctacaaatcaaag gggcccttaa gttcgttagc ttttctgttt gaaacatgtt 3120 ttgcacggattatttagttg attaacacga taaatcaata ttttaagaaa taaactttac 3180 acataatttagagtaagtaa tctaacaatg cagtagaagt tttttttttt ttgagaaatc 3240 gtattttaataagagtggag caaaaacctt tggcaaaaat ttggagaaaa gaagctcaaa 3300 aaacaaaatccgtggcgaac cggcgattgt ggtattagta ctgcaaagaa tacgaaaaag 3360 accaaaattgttgcctgaat tcagccgtag tttacccttg gagtacgtac atggttcaat 3420 taatttgctctagctgataa ttgtgcttga tgcctgcaga tgatgcggag aagaaaccca 3480 aaagtgtagttgatgttggc tgtggcattg gtggtagctc aagatacttg gcgaacaaat 3540 acggagcgcaatgctacggc atcacgttga gtccggtgca ggctgaaaga ggaaatgccc 3600 tcgcggcagagcaagggtta tcagacaagg tgcgtattac tactgtttat tctgttctaa 3660 aaaaaattctactgtttatt cgtatcggga tttaatctcg ctgtagctag tcatagttac 3720 atttgacaatatcagagggt ttaagctctg attactcact gctggtgtga cgacaatctg 3780 tttaagcaggtctcctttca agttggtgat gcattggagc agccttttcc tgatgggcag 3840 tttgatcttgtctggtccat ggagagtggc gagcacatgc cagacaaacg gcaggtaaga 3900 tactcctcttttttatcctt acagaaaaaa agaaaatggg aaaatgtaat ccctccgttt 3960 caggttataagactttctag cattgccacg ttcatataga tgttaatgaa tctaggcaca 4020 catatatgtttagattcatt aacatatata tgaatatggg taatactaga aagttttata 4080 atatgaaacggaagaaatat cttggatcgg agaacgtttc aaatctagcc atcaggttac 4140 ggtggcatgggttaccatgc atcggttgga atcttggttg agcaatcgca tctgtcgatt 4200 ttaatttgccgggttgcgaa aatgtagaaa aacgagagga tattaatgaa acaattctgc 4260 tatttattaaaatcagatgt caatcaactc atcttgagag gagtctgtat tggatgttac 4320 tgaatagtttttggatcttt tcactctctt tttttagcta agagttctta cctgagtctt 4380 ttacagtttgtaagcgagct ggcacgcgtc gcagctcctg gggcgagaat aatcattgtg 4440 acctggtgccataggaacct cgagccatcc gaagagtccc tgaaacctga tgagctgaat 4500 ctcctgaaaaggatatgcga tgcatattat ctcccagact ggtgctctcc ttctgattat 4560 gtcaaaattgccgagtcact gtctcttgag gtaaaaaaac ttttcatgct ctgaactcgt 4620 aagtgaatttaagttacaac ttgatatggt ttgcacatca acttgcgtac catgccgatt 4680 tgcattctcgcaagagattc ttgcatgtgt gtgacatgtg aaatgtgcca ggatataagg 4740 acagctgattggtcagagaa cgtcgcccca ttctggcctg cggttataaa atcagcattg 4800 acatggaaaggtttaacttc tctgctaaga agtggtatga tcttgccatc ttcctttcct 4860 ccacttatgattatcggcaa acagatgttg gacaaaactg aactaatttg tgttggcttc 4920 gtcttaatttgaagggtgga agacgataag aggtgcaatg gtgatgcctc tgatgatcga 4980 aggatacaagaaagggctca tcaaattcac catcatcacc tgtcgcaagc ccgaaacaac 5040 gcagtagtaccctagtagtg aaattacgct cctgctatct tctccatcac gaataatgca 5100 aattctgacgagttagcacc tactgatggc gatttgttga tttggggaac agccagtgca 5160 ctgttaccacgtcattgatt ttgtactcgt cagacttaaa aaaaaaatat ccatgaatgt 5220 gcactccaaatacgtcaaga aattcttaga tcttcagacc aactcgtcag ctagaggtgg 5280 ctaaaaagctcatttagctc cctcggtgca agattgcttt gattgaccta gttttcttct 5340 taaaactaaaactattttac tttggatgga agttagtttc actctgtctg ggctcggctc 5400 attagctcgttaagaaaaac agtttcaaat gataaaataa cataataaat caatttcgaa 5460 gaaaaatggagtagataaaa agcacagccg ggctcaaccg agggcttatt tagattgaag 5520 gattttcatgggaattttgg aggactcaaa tccttgataa aatttctaca agcccctttg 5580 gaacataacaggatcgctat aaatcctatg cctcccaatt tcataggaaa ataaacatga 5640 gctcaaactcatgtttttat ttccctttga gaaatctaat gcactctctc cctatctctc 5700 tccctttgagaagtctaatg cactcgctcc ctatctctat agtttctctc ctttgaaatt 5760 tctgtgtttacttgctacta atcatccaaa ggcaactact ctatagtatt catgtatttt 5820 aaatttctgtaggattttat agagcgtggc atcttatttc tatgtttttt ttatcactgt 5880 gtttttaaaattttgcgctc caaaggcgca ctagcagtgc agcagcgtca ctatgtcagc 5940 tcgacgccgaaccgccatcc ttctccgatg gcgcggcgcg ctcaccacgc cacccgcgcc 6000 gcacggtgaaccaaagctgg cacggcacgg gcaggcacca gcacagtcgg gcaaccgacg 6060 gtctgccgcgcgccgcgtgg tgctccggtc acggagacgt cggcaatcgg cgtccatcga 6120 tgttgccgtctcactctcgt cgtccttcag gatgacccac ttgcccgctc cataattcac 6180 aaaaatagccaagcaaaaga acggctccat ctggtacgaa aatgacaggg ccctggttaa 6240 gaaatcaacgacgttgcgaa gaaagcttgg tctctgacga gagaatggca ggaaacgata 6300 aggtagtcaggtagagctag aactgtcagt ttcacacagg atcattcact ttctctccag 6360 cagctcaacaaaggctcgcc aaatcacatc aacaagaatg cttattgtat aatcatccct 6420 cgtgttcaccaataaatctg ctgcggctta ttggtattgt atgactaaga acagggttcg 6480 tggctatagctgacatccgt aaatttgtaa tccatcttgg aattaacaaa aaggtagcac 6540 acaatttgagcccaatctct gcttaatttg tcggcaggac gtccaacttt tatcagttct 6600 tcacggtggttacatgccta agaagcatct ggatcacttc aggggagaca tttgcagcct 6660 gctccttcagttgcgcaatt ttcgtgtcag tttcttgctc aagccgtttt acgtttgcac 6720 cagaatcaccgctactctgg aaagaaacag gaaggttagc ggcaatagct cttctaattc 6780 cacgaactatacaagattga aagatgcata cctccgcaac cttcctctgg aactcagctt 6840 ccatctgagctgatgaacta ctgt 6864 51 32 DNA Artificial Sequence Synthetic Primer 51gcggccgcac tttcttgttc cgccaacctc tc 32 52 30 DNA Artificial SequenceSynthetic Primer 52 cctgcaggcg ctgaaaagca cttaaaagac 30 53 64 DNAArtificial Sequence Synthetic Primer 53 ggggacaagt ttgtacaaaa aagcaggctgcggccgcaca atgaaagcga cactcgcacc 60 accc 64 54 59 DNA ArtificialSequence Synthetic Primer 54 ggggaccact ttgtacaaga aagctgggtc ctgcaggttatagaggcttc tggcaagtg 59 55 63 DNA Artificial Sequence Synthetic Primer55 ggggacaagt ttgtacaaaa aagcaggctg cggccgcaca atgccgataa catctatttc 60cgc 63 56 59 DNA Artificial Sequence Synthetic Primer 56 ggggaccactttgtacaaga aagctgggtc ctgcaggcta ttcgggctta cggcatgtg 59 57 62 DNAArtificial Sequence Synthetic Primer 57 ggggacaagt ttgtacaaaa aagcaggctgcggccgcaca atggctgccg cgttacaatt 60 ac 62 58 57 DNA Artificial SequenceSynthetic Primer 58 ggggaccact ttgtacaaga aagctgggtc ctgcaggctactcagctggc ttccggc 57 59 73 DNA Artificial Sequence Synthetic Primer 59ggggacaagt ttgtacaaaa aagcaggctt agaaggagat agaaccatgg tggctgtgac 60ggctgctgct acc 73 60 59 DNA Artificial Sequence Synthetic Primer 60ggggaccact ttgtacaaga aagctgggtc ctgcaggtta gagaggcttc tggcaagtg 59 6172 DNA Artificial Sequence Synthetic Primer 61 ggggacaagt ttgtacaaaaaagcaggctt agaaggagat agaaccatgg ctactgccga 60 caagactcag ag 72 62 59DNA Artificial Sequence Synthetic Primer 62 ggggaccact ttgtacaagaaagctgggtc ctgcaggcta ttcgggctta cggcatgtg 59 63 73 DNA ArtificialSequence Synthetic Primer 63 ggggacaagt ttgtacaaaa aagcaggctt agaaggagatagaaccatgg tgaaggcggc 60 ggcgtcgtct ttg 73 64 57 DNA Artificial SequenceSynthetic Primer 64 ggggaccact ttgtacaaga aagctgggtc ctgcaggctactcagctggc ttccggc 57 65 73 DNA Artificial Sequence Synthetic Primer 65ggggacaagt ttgtacaaaa aagcaggctt agaaggagat agaaccatgg cccttagcgt 60ggtcgcggcc gag 73 66 54 DNA Artificial Sequence Synthetic Primer 66ggggaccact ttgtacaaga agctgggtct tattcaggct ttttgcatgt gatg 54 67 74 DNAArtificial Sequence Synthetic Primer 67 ggggacaagt ttgtacaaaa aagcaggcttagaaggagat agaaccatga gtgcaacact 60 ttaccagcaa attc 74 68 56 DNAArtificial Sequence Synthetic Primer 68 ggggaccact ttgtacaaga aagctgggtcctactactta ttgccgcaca gtaagc 56 69 76 DNA Artificial Sequence SyntheticPrimer 69 ggggacaagt ttgtacaaaa aagcaggctt agaaggagat agaaccatgagtgcaacact 60 ttaccaacaa attcag 76 70 56 DNA Artificial SequenceSynthetic Primer 70 ggggaccact ttgtacaaga aagctgggtc ctatcacttatccccacaaa gcaacc 56 71 71 DNA Artificial Sequence Synthetic Primer 71ggggacaagt ttgtacaaaa aagcaggctt agaaggagat agaaccatga gttggttgtt 60ttctacactg g 71 72 56 DNA Artificial Sequence Synthetic Primer 72ggggaccact ttgtacaaga aagctgggtc ctattacttt tgagcaacct tgatcg 56 73 71DNA Artificial Sequence Synthetic Primer 73 ggggacaagt ttgtacaaaaaagcaggctt agaaggagat agaaccatgg cctcgtcgac 60 ggctcaggcc c 71 74 62 DNAArtificial Sequence Synthetic Primer 74 ggggaccact ttgtacaaga aagctgggtcctgcaggcta cgcggctcca ggcttgcgac 60 ag 62 75 31 DNA Artificial SequenceSynthetic Primer 75 catgccatgg gaatgaaagc aactctagca g 31 76 33 DNAArtificial Sequence Synthetic Primer 76 gtcagaattc ttattagagt ggcttctggcaag 33 77 37 DNA Artificial Sequence Synthetic Primer 77 gcggccgcacaatgaaagca actctagcag caccctc 37 78 33 DNA Artificial Sequence SyntheticPrimer 78 cctgcaggtt agagtggctt ctggcaagtg atg 33 79 24 DNA ArtificialSequence Synthetic Primer 79 ccacgtgagc tccttcctct tccc 24 80 33 DNAArtificial Sequence Synthetic Primer 80 gtgccatggc agatctgatg atggattgatgga 33 81 51 DNA Artificial Sequence Synthetic Primer 81 gagtgatggttaatgcatga atgcatgatc agatctgcca tggtccgtcc t 51 82 51 DNA ArtificialSequence Synthetic Primer 82 gagtgatggt taatccatca atccatcatc agatctgccatggtccgtcc t 51 83 71 DNA Artificial Sequence Synthetic Primer 83ggggacaagt ttgtacaaaa aagcaggctt agaaggagat agaaccatga gttggttgtt 60ttctacactg g 71 84 56 DNA Artificial Sequence Synthetic Primer 84ggggaccact ttgtacaaga aagctgggtc ctattacttt tgagcaacct tgatcg 56 85 1363DNA Zea mays 85 aacagtgccg cggtgcgcgc acacacagcc accacccccc cgtcccctcgcctcggcctc 60 ttttaaatat cgcgcatccc ggcgccgcaa atggctcacg cggcgctgctccattgctcc 120 cagtcctcca ggagcctcgc agcctgccgc cgcggcagcc actaccgcgccccttcgcac 180 gtcccgcgcc actcccgccg tctccgacgc gccgtcgtca gcctgcgtccgatggcctcg 240 tcgacggctc aggcccccgc gacggcgccg ccgggtctga aggagggcatcgcggggctg 300 tacgacgagt cgtcggggct gtgggagaac atctggggcg accacatgcaccacggcttc 360 tacgactcga gcgaggccgc ctccatggcc gatcaccgcc gcgcccagatccgcatgatc 420 gaggaggcgc tcgccttcgc cggtgtccca gcctcagatg atccagagaagacaccaaaa 480 acaatagtcg atgtcggatg tggcattggt ggtagctcaa ggtacttggcgaagaaatac 540 ggagcgcagt gcactgggat cacgttgagc cctgttcaag ccgagagaggaaatgctctc 600 gctgcagcgc aggggttgtc ggatcaggtt actctgcaag ttgctgatgctctggagcaa 660 ccgtttcctg acgggcagtt cgatctggtg tggtccatgg agagtggcgagcacatgccg 720 gacaagagaa agtttgttag tgagctagca cgcgtggcgg ctcctggagggacaataatc 780 atcgtgacat ggtgccatag gaacctggat ccatccgaaa cctcgctaaagcccgatgaa 840 ctgagcctcc tgaggaggat atgcgacgcg tactacctcc cggactggtgctcaccttca 900 gactatgtga acattgccaa gtcactgtct ctcgaggata tcaagacagctgactggtcg 960 gagaacgtgg ccccgttttg gcccgccgtg ataaaatcag cgctaacatggaagggcttc 1020 acctctctgc tgacgaccgg atggaagacg atcagaggcg cgatggtgatgccgctaatg 1080 atccagggct acaagaaggg gctcatcaaa ttcaccatca tcacctgtcgcaagcctgga 1140 gccgcgtagg aggaggccaa ggagcacaag ttactagcac aggcacaggagtgccaagtg 1200 caataatgta gatccgtggc cccatcgccg tctactcatc tatactgcaccaaaatcaac 1260 attctcctag gacatgttaa ataattttct gccactcgtc gagatatttcaaattcactg 1320 ttccacaaaa aaaaaaaagg cgccgccgac tagtgagctg tcg 1363

What is claimed is:
 1. A substantially purified nucleic acid moleculecomprising a nucleic acid sequence selected from the group consisting ofSEQ ID NOs: 2-17, 50, and
 85. 2. A substantially purified nucleic acidmolecule comprising a nucleic acid sequence that encodes a polypeptidemolecule comprising a sequence selected from the group consisting of SEQID NOs: 19-31 and 33-38.
 3. A substantially purified nucleic acidmolecule comprising as operably linked components: (A) a promoter regionwhich functions in a plant cell to cause the production of an mRNAmolecule; (B) a heterologous nucleic acid molecule encoding apolypeptide molecule comprising a sequence selected from the groupconsisting of SEQ ID NOs: 19-31 and 33-38.
 4. The nucleic acid moleculeof claim 3, further comprising a 3′ non-translated sequence thatfunctions in said plant cell to cause termination of transcription andaddition of polyadenylated ribonucleotides to a 3′ end of the mRNAmolecule.
 5. A substantially purified protein comprising an amino acidsequence selected from the group consisting of SEQ ID NOs: 19-31 and33-38.
 6. An antibody capable of specifically binding a substantiallypurified protein with an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 19-31 and 33-38.
 7. A transformed plantcomprising an exogenous nucleic acid molecule comprising a nucleic acidsequence selected from the group consisting of SEQ ID NOs: 2-17, 50, and85 and complements thereof.
 8. The transformed plant according to claim7, wherein said plant is selected from the group consisting of canola,corn, Brassica campestris, Brassica napus, oilseed rape, rapeseed,soybean, crambe, mustard, castor bean, peanut, sesame, cottonseed,linseed, safflower, oil palm, flax and sunflower.
 9. The transformedplant according to claim 8, wherein said plant is canola or oilseedrape.
 10. The transformed plant according to claim 8, wherein said plantis soybean or soybean line A3244.
 11. The transformed plant according toclaim 7, wherein said transformed plant produces a seed with increasedα-tocopherol levels relative to a plant with a similar geneticbackground but lacking said exogenous nucleic acid molecule.
 12. Thetransformed plant according to claim 11, wherein α-tocopherol is thepredominant species of tocopherol in said seed
 13. The transformed plantof claim 11, wherein said α-tocopherol species comprises greater thanabout 90% of the total tocopherol content of said seed.
 14. Thetransformed plant according to claim 7, wherein said nucleic acidmolecule is operably linked to a promoter.
 15. The transformed plantaccording to claim 14, wherein said promoter is a seed specificpromoter.
 16. The transformed plant according to claim 15, wherein saidpromoter is the p7S promoter.
 17. The transformed plant according toclaim 15, wherein said promoter is the Arcelin 5 promoter or mutantsthereof.
 18. The transformed plant according to claim 17, wherein saidpromoter comprises the nucleic acid molecule of SEQ ID NO:
 81. 19. Thetransformed plant according to claim 17, wherein said promoter comprisesthe nucleic acid molecule of SEQ ID NO:
 82. 20. A transformed soybeanline A3244 plant, comprising an exogenous nucleic acid moleculecomprising a nucleic acid sequence of SEQ ID NO: 2, operably linked to anucleic acid molecule comprising a nucleotide sequence selected from thegroup consisting of p7S, SEQ ID NO: 81, and SEQ ID NO:
 82. 21. Atransformed plant having an exogenous nucleic acid molecule comprising anucleic acid sequence selected from the group consisting of SEQ ID NOs:2-17, 50, and 85 and complements thereof, wherein said transformed plantproduces a seed with increased α-tocotrienol levels relative to a plantwith a similar genetic background but lacking said exogenous nucleicacid molecule.
 22. A transformed plant having an exogenous nucleic acidmolecule that encodes a polypeptide molecule comprising an amino acidsequence selected from the group consisting of SEQ ID NOs: 19-31 and33-40.
 23. The transformed plant according to claim 22, wherein saidplant is selected from the group consisting of canola, rapeseed, corn,Brassica campestris, Brassica napus, oilseed rape, soybean, crambe,mustard, castor bean, peanut, sesame, cottonseed, linseed, safflower,oil palm, flax and sunflower.
 24. The transformed plant according toclaim 23, wherein said plant is canola or oilseed rape.
 25. Thetransformed plant according to claim 23, wherein said plant is soybean.26. The transformed plant according to claim 20, wherein saidtransformed plant produces a seed with increased α-tocopherol levelsrelative to a plant with a similar genetic background but lacking saidexogenous nucleic acid molecule.
 27. The transformed plant according toclaim 26, wherein α-tocopherol is the predominant species of tocopherolin said seed
 28. The transformed plant of claim 26, wherein saidα-tocopherol species comprises greater than about 90% of the totaltocopherol content of said seed.
 29. The transformed plant according toclaim 20, wherein said nucleic acid molecule is operably linked to apromoter.
 30. The transformed plant according to claim 29, wherein saidpromoter is a seed specific promoter.
 31. A transformed plant having anexogenous nucleic acid molecule that encodes a polypeptide moleculecomprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 19-31 and 33-40, wherein said transformed plant produces aseed with increased α-tocotrienol levels relative to a plant with asimilar genetic background but lacking said exogenous nucleic acidmolecule.
 32. A transformed plant having an exogenous nucleic acidmolecule that encodes a polypeptide molecule comprising an amino acidsequence selected from the group consisting of SEQ ID NOs: 46-49. 33.The transformed plant according to claim 32, wherein said plant isselected from the group consisting of canola, rapeseed, corn, Brassicacampestris, Brassica napus, oilseed rape, soybean, crambe, mustard,castor bean, peanut, sesame, cottonseed, linseed, safflower, oil palm,flax and sunflower.
 34. The transformed plant according to claim 33,wherein said plant is canola or oilseed rape.
 35. The transformed plantaccording to claim 33, wherein said plant is soybean.
 36. Thetransformed plant according to claim 32, wherein said transformed plantproduces a seed with decreased δ-tocopherol and β-tocopherol levelsrelative to a plant with a similar genetic background but lacking saidexogenous nucleic acid molecule.
 37. The transformed plant according toclaim 36, wherein γ-tocopherol is the predominant species of tocopherolin said seed
 38. The transformed plant of claim 36, wherein saidδ-tocopherol species comprises greater than about 90% of the totaltocopherol content of said seed.
 39. The transformed plant according toclaim 32, wherein said nucleic acid molecule is operably linked to apromoter.
 40. The transformed plant according to claim 39, wherein saidpromoter is a seed specific promoter.
 41. The transformed plant of claim32, wherein said transformed plant produces a seed with decreasedδ-tocotrienol and β-tocotrienol levels relative to a plant with asimilar genetic background but lacking said exogenous nucleic acidmolecule.
 42. A method for reducing expression of MT1 or GMT in a plantcomprising: (A) transforming a plant with a nucleic acid molecule, saidnucleic acid molecule having an exogenous promoter region whichfunctions in plant cells to cause the production of a mRNA molecule,wherein said exogenous promoter region is linked to a transcribednucleic acid molecule having a transcribed strand and a non-transcribedstrand, wherein said transcribed strand is complementary to a nucleicacid molecule comprising a nucleic acid sequence selected from the groupconsisting of: SEQ ID NOs: 2-17, 42-45, 50, and 85; and wherein saidtranscribed nucleic acid molecule is linked to a 3′ non-translatedsequence that functions in the plant cells to cause termination oftranscription and addition of polyadenylated ribonucleotides to a 3′ endof the mRNA sequence; and (B) growing said transformed plant.
 43. Atransformed plant comprising a nucleic acid molecule comprising anexogenous promoter region which functions in plant cells to cause theproduction of a mRNA molecule, wherein said exogenous promoter region islinked to a transcribed nucleic acid molecule having a transcribedstrand and a non-transcribed strand, wherein said transcribed strand iscomplementary to a nucleic acid molecule comprising a nucleic acidsequence selected from the group consisting of SEQ ID NOs: 2-17, 42-45,50, and 85, and wherein said transcribed nucleic acid molecule is linkedto a 3′ non-translated sequence that functions in the plant cells tocause termination of transcription and addition of polyadenylatedribonucleotides to a 3′ end of the mRNA sequence.
 44. The transformedplant of claim 43, wherein the expression of MT1 is reduced relative toa plant with a similar genetic background but lacking said exogenousnucleic acid molecule.
 45. The transformed plant of claim 43, whereinthe expression of GMT is reduced relative to a plant with a similargenetic background but lacking said exogenous nucleic acid molecule. 46.A method for increasing the γ-tocopherol content in a plant comprising:(A) transforming a plant with a nucleic acid molecule, said nucleic acidmolecule having an exogenous promoter region which functions in plantcells to cause the production of a mRNA molecule, wherein said exogenouspromoter region is linked to a transcribed nucleic acid molecule havinga transcribed strand and a non-transcribed strand, wherein saidtranscribed strand is complementary to a nucleic acid moleculecomprising a nucleic acid sequence selected from the group consisting ofSEQ ID NOs: 2-17, 50, and 85; and wherein said transcribed nucleic acidmolecule is linked to a 3′ non-translated sequence that functions in theplant cells to cause termination of transcription and addition ofpolyadenylated ribonucleotides to a 3′ end of the mRNA sequence; and (B)growing said transformed plant.
 47. A transformed plant comprising: (A)a first nucleic acid molecule comprising an exogenous promoter regionwhich functions in plant cells to cause the production of a mRNAmolecule, wherein said exogenous promoter region is linked to atranscribed nucleic acid molecule having a transcribed strand and anon-transcribed strand, wherein said transcribed strand is complementaryto a nucleic acid molecule comprising a nucleic acid sequence selectedfrom the group consisting of SEQ ID NOs: 2-17, 50, and 85, and whereinsaid transcribed nucleic acid molecule is linked to a 3′ non-translatedsequence that functions in the plant cells to cause termination oftranscription and addition of polyadenylated ribonucleotides to a 3′ endof the mRNA sequence; and (B) a second nucleic acid molecule comprisingan exogenous promoter region which functions in plant cells to cause theproduction of a mRNA molecule, wherein said exogenous promoter region islinked to a nucleic acid molecule comprising a sequence selected fromthe group consisting of SEQ ID NOs: 42-45.
 48. The transformed plant ofclaim 47, wherein the γ-tocopherol content of said transformed plant isincreased relative to a plant with a similar genetic background butlacking said exogenous nucleic acid molecule.
 49. A method of producinga plant having a seed with an increased α-tocopherol level comprising:(A) transforming said plant with a nucleic acid molecule, wherein saidnucleic acid molecule comprises a nucleic acid sequence selected fromthe group consisting of SEQ ID NOs: 2-17, 50, and 85; and (B) growingsaid transformed plant.
 50. The method according to claim 49, whereinsaid plant is selected from the group consisting of canola, rapeseed,corn, Brassica campestris, Brassica napus, oilseed rape, soybean,crambe, mustard, castor bean, peanut, sesame, cottonseed, linseed,safflower, oil palm, flax and sunflower.
 51. The method according toclaim 50, wherein said plant is canola or oilseed rape.
 52. The methodaccording to claim 50, wherein said plant is soybean.
 53. The methodaccording to claim 49, wherein α-tocopherol is the predominant speciesof tocopherol in said seed.
 54. The method according to claim 49,wherein α-tocopherol comprises greater than about 90% of the totaltocopherol content of said seed.
 55. A method of producing a planthaving a seed with an increased α-tocotrienol level comprising: (A)transforming said plant with a nucleic acid molecule, wherein saidnucleic acid molecule comprises a nucleic acid sequence selected fromthe group consisting of SEQ ID NOs: 2-17, 50, and 85; and (B) growingsaid transformed plant.
 56. A method of producing a plant having a seedwith an increased α-tocopherol level comprising: (A) transforming saidplant with a nucleic acid molecule, wherein said nucleic acid moleculecomprises a sequence encoding an polypeptide molecule comprising anamino acid sequence selected from the group consisting of SEQ ID NOs:19-31 and 33-40; and (B) growing said transformed plant.
 57. The methodaccording to claim 56, wherein said plant is selected from the groupconsisting of canola, corn, Brassica campestris, Brassica napus, oilseedrape, soybean, crambe, mustard, castor bean, peanut, sesame, cottonseed,linseed, safflower, oil palm, flax and sunflower.
 58. The methodaccording to claim 57, wherein said transformed plant is canola oroilseed rape.
 59. The method according to claim 57, wherein saidtransformed plant is soybean.
 60. A method of producing a plant having aseed with an increased α-tocotrienol level comprising: (A) transformingsaid plant with a nucleic acid molecule, wherein said nucleic acidmolecule comprises a sequence encoding an polypeptide moleculecomprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 19-31 and 33-40; and (B) growing said transformed plant. 61.A method of producing a plant having a seed with an increasedγ-tocopherol level comprising: (A) transforming said plant with anucleic acid molecule, wherein said nucleic acid molecule comprises anucleic acid sequence that encodes an polypeptide molecule comprising asequence selected from the group consisting of SEQ ID NOs: 46-49 and (B)growing said transformed plant.
 62. A method of producing a plant havinga seed with an increased γ-tocopherol level comprising: (A) transformingsaid plant with a nucleic acid molecule, wherein said nucleic acidmolecule comprises a nucleic acid sequence selected from the groupconsisting of SEQ ID NOs: 42-45 and (B) growing said transformed plant.63. The method according to claim 62, wherein said plant is selectedfrom the group consisting of canola, corn, Brassica campestris, Brassicanapus, oilseed rape, soybean, crambe, mustard, castor bean, peanut,sesame, cottonseed, linseed, safflower, oil palm, flax and sunflower.64. The method according to claim 63, wherein said transformed plant iscanola or oilseed rape.
 65. The method according to claim 63, whereinsaid transformed plant is soybean.
 66. The method according to claim 62,wherein γ-tocopherol is the predominant species of tocopherol in saidseed.
 67. The method according to claim 62, wherein said γ-tocopherolcomprises greater than about 90% of the total tocopherol content of saidseed.
 68. A method of producing a plant having a seed with an increasedγ-tocotrienol level comprising: (A) transforming said plant with anucleic acid molecule, wherein said nucleic acid molecule comprises anucleic acid sequence that encodes a polypeptide molecule comprising asequence selected from the group consisting of SEQ ID NO: 47 through SEQID NO: 49; and (B) growing said transformed plant.
 69. A method ofproducing a plant having a seed with an increased γ-tocotrienol levelcomprising: (A) transforming said plant with a nucleic acid molecule,wherein said nucleic acid molecule comprises a nucleic acid sequenceselected from the group consisting of SEQ ID NOs: 42-46; and (B) growingsaid transformed plant.
 70. A seed derived from a transformed planthaving an exogenous nucleic acid molecule comprising a nucleic acidsequence selected from the group consisting of SEQ ID NOs: 2-17, 50, and85, wherein said seed has an increased α-tocopherol level relative to aseed from a plant having a similar genetic background but lacking saidexogenous nucleic acid molecule.
 71. The seed of claim 70, whereinα-tocopherol is the predominant species of tocopherol in said seed. 72.The seed of claim 70, wherein α-tocopherol comprises greater than about90% of the total tocopherol content of said seed.
 73. A seed derivedfrom a transformed plant having an exogenous nucleic acid moleculecomprising a nucleic acid sequence selected from the group consisting ofSEQ ID NOs: 2-17, 50, and 85, wherein said seed has an increasedα-tocotrienol level relative to a seed from a plant having a similargenetic background but lacking said exogenous nucleic acid molecule. 74.Oil derived from a seed of a transformed plant, wherein said transformedplant comprises an exogenous nucleic acid molecule comprising a sequenceselected from the group consisting of SEQ ID NOs: 2-17, 42-45, 50, and85.
 75. Feedstock comprising a transformed plant or part thereof,wherein said transformed plant has an exogenous nucleic acid moleculecomprising a nucleic acid sequence selected from the group consisting ofSEQ ID NOs: 2-17, 42-45, 50, and
 85. 76. The feedstock of claim 75,wherein the nucleic acid comprises a sequence selected from the groupconsisting of SEQ ID NOs: 2-17, 50 and 85 wherein said plant producesseeds with increased α-tocopherol levels relative to a plant with asimilar genetic background but lacking said exogenous nucleic acidmolecule.
 77. A meal comprising plant material manufactured from atransformed plant, wherein said transformed plant has an exogenousnucleic acid molecule comprising a nucleic acid sequence selected fromthe group consisting of SEQ ID NOs: 2-17, 42-45, 50, and
 85. 78. A seedderived from a transformed plant comprising an exogenous nucleic acidmolecule encoding a polypeptide molecule comprising a sequence selectedfrom the group consisting of SEQ ID NOs: 19-31 and 33-40 wherein saidseed has an increased α-tocopherol level relative to seeds from a planthaving a similar genetic background but lacking said exogenous nucleicacid molecule.
 79. A seed derived from a transformed plant comprising anexogenous nucleic acid molecule encoding a polypeptide moleculecomprising a sequence selected from the group consisting of SEQ ID NOs:19-31 and 33-40 wherein said seed has an increased α-tocotrienol levelrelative to seeds from a plant having a similar genetic background butlacking said exogenous nucleic acid molecule.
 80. The seed of claim 79,wherein α-tocopherol is the predominant species of tocopherol in saidseed.
 81. The seed of claim 79, wherein α-tocopherol comprises greaterthan about 90% of the total tocopherol content of said seed.
 82. Oilderived from a seed of a transformed plant, wherein said transformedplant comprises an exogenous nucleic acid molecule encoding apolypeptide molecule comprising a sequence selected from the groupconsisting of SEQ ID NOs: 19-31, 33-40, and 46-49.
 83. Feedstockcomprising a transformed plant or part thereof, wherein said transformedplant comprises an exogenous nucleic acid molecule encoding apolypeptide molecule comprising a sequence selected from the groupconsisting of SEQ ID NOs: 19-31 and 33-40.
 84. The feedstock of claim83, wherein said plant produces seeds with increased α-tocopherol levelsrelative to a plant with a similar genetic background but lacking saidexogenous nucleic acid molecule.
 85. A meal comprising plant materialmanufactured from a transformed plant, wherein said transformed plantcomprises an exogenous nucleic acid molecule encoding a polypeptidemolecule comprising a sequence selected from the group consisting of SEQID NOs: 19-31, 33-40, and 46-49.
 86. A seed derived from a transformedplant comprising an exogenous nucleic acid molecule encoding apolypeptide molecule comprising a sequence selected from the groupconsisting of SEQ ID NOs: 46-49, wherein said seed has an increased γtocopherol level relative to seeds from a plant having a similar geneticbackground but lacking said exogenous nucleic acid molecule.
 87. A seedderived from a transformed plant having an exogenous nucleic acidmolecule encoding a polypeptide molecule comprising a sequence selectedfrom the group consisting of SEQ ID NOs: 46-49, wherein said seed has anincreased γ tocotrienol level relative to seeds from a plant having asimilar genetic background but lacking said exogenous nucleic acidmolecule.
 88. Feedstock comprising a transformed plant or part thereof,wherein said transformed plant comprises an exogenous nucleic acidmolecule encoding a polypeptide molecule comprising a sequence selectedfrom the group consisting of SEQ ID NOs: 46-49.
 89. Meal comprisingplant material manufactured from a transformed plant, wherein saidtransformed plant has an exogenous nucleic acid molecule encoding anamino acid sequence selected from the group consisting of SEQ ID NOs:46-49.
 90. A host cell comprising a nucleic acid molecule comprising anucleic acid sequence selected from the group consisting of SEQ ID NOs:2-17, 42-45, 50, and 85 and complements thereof.
 91. The host cellaccording to claim 90, wherein said cell is a bacterial cell.
 92. Thehost cell according to claim 90, wherein said cell is an Agrobacteriumtumefaciens or E. coli cell.
 93. A transformed plant comprising anintroduced first nucleic acid molecule that encodes a polypeptidemolecule comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 19-31, 33-38, and 39-41, and an introducedsecond nucleic acid molecule encoding an enzyme selected from the groupconsisting of tyrA, slr1736, ATPT2, dxs, dxr, GGPPS, HPPD, GMT, MT1,tMT2, AANT1, slr 1737, and an antisense construct for homogentisic aciddioxygenase.
 94. A transformed plant comprising an introduced firstnucleic acid molecule that encodes a polypeptide molecule comprising anamino acid sequence selected from the group consisting of SEQ ID NOs:46-49, and an introduced second nucleic acid molecule encoding an enzymeselected from the group consisting of tyrA, slr1736, ATPT2, dxs, dxr,GGPPS, HPPD, GMT, MT1, tMT2, AANT1, slr 1737, and an antisense constructfor homogentisic acid dioxygenase.
 95. A plant comprising an introducednucleic acid molecule comprising a nucleic acid sequence selected fromthe group consisting of 42-45, wherein said transformed plant produces aseed having increased total tocopherol relative to seed of a plant witha similar genetic background but lacking said introduced nucleic acidmolecule.
 96. A plant comprising an introduced nucleic acid moleculecomprising a nucleic acid sequence selected from the group consisting of2-17, 50, 85, wherein said transformed plant produces a seed havingincreased total tocopherol relative to seed of a plant with a similargenetic background but lacking said introduced nucleic acid molecule.97. A plant comprising a first introduced nucleic acid moleculecomprising a nucleic acid sequence selected from the group consisting of2-17, 50, and 85 and a second introduced nucleic acid moleculecomprising a nucleic acid sequence selected from the group consisting of42-45, wherein said transformed plant produces a seed having increasedtotal tocopherol relative to seed of a plant with a similar geneticbackground but lacking both said introduced first nucleic acid moleculeand said introduced second nucleic acid molecule.